Vaccine candidates for human respiratory syncytial virus (rsv) having attenuated phenotypes

ABSTRACT

Reported herein are presumptively de-attenuating mutations that are useful, either individually or in combinations that may include other known mutations, in producing recombinant strains of human respiratory syncytial virus (RSV) exhibiting attenuation phenotypes. Also described herein is a novel RSV construct, Min_L-NPM2-1(N88K)L, which exhibits an attenuated phenotype, is stable and is as immunogenic as wild type RSV. The recombinant RSV strains described here are suitable for use as live-attenuated RSV vaccines. Exemplary vaccine candidates are described. Also provided are polynucleotide sequences capable of encoding the described viruses, as well as methods for producing and using the viruses.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.17/008,025, filed Aug. 31, 2020, which is a continuation of U.S. patentapplication Ser. No. 16/335,099, filed Mar. 20, 2019, now U.S. Pat. No.10,808,012, issued Oct. 20, 2020, which is a national stage applicationunder 35 U.S.C. 371 of PCT Application No. PCT/US2017/053047 having aninternational filing date of Sep. 22, 2017, which designated the UnitedStates, which PCT application claimed priority to U.S. ProvisionalApplication Ser. No. 62/399,133, filed Sep. 23, 2016, and U.S.Provisional Application Ser. No. 62/400,476, filed Sep. 27, 2016, thecontents of each of which are incorporated herein by reference in theirentireties for all purposes.

GOVERNMENT RIGHTS

The Government of the United States has certain rights in thisinvention.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted as an electronictext file named “Sequence_Listing_3000094-009004_ST25.txt”, having asize in bytes of 99,901 bytes, and created on Jun. 27, 2022. Theinformation contained in this electronic file is hereby incorporated byreference in its entirety pursuant to 37 CFR § 1.52(e)(5).

FIELD OF THE INVENTION

The subject matter disclosed herein relates to respiratory syncytialvirus (RSV) and attenuated, mutant strains thereof suitable for use asvaccines.

BACKGROUND OF THE INVENTION

Human respiratory syncytial virus (RSV) infects nearly everyoneworldwide early in life and is responsible for considerable mortalityand morbidity (for general reviews, see: Collins and Graham, 2008, JVirol. 82:2040-2055; Collins and Melero, 2011, Virus Res 162: 80-99;Collins and Karron, 2013, Fields Virology 6th Edition, pp 1086-1123;Collins, et al., 2013, Curr Top Microbiol Immunol 372:3-38). In theUnited States alone, RSV is responsible for 75,000-125,000hospitalizations yearly, and conservative estimates indicate that RSV isresponsible worldwide for 64 million pediatric infections and 160,000 ormore pediatric deaths each year. Another notable feature of RSV is thatsevere infection in infancy frequently is followed by lingering airwaydysfunction, including a predisposition to airway reactivity, that insome individuals lasts for years and can extend into adolescence andbeyond. RSV infection exacerbates asthma and may be involved ininitiating asthma.

RSV is a negative strand RNA virus of the pneumoviridae family. Thegenome of RSV is a single, negative-sense strand of RNA of 15.2kilobases that is transcribed by the viral polymerase into 10 mRNAs by asequential stop-start mechanism that initiates at a single viralpromoter at the 3′ end of the genome. Each mRNA encodes a single majorprotein, with the exception of the M2 mRNA that has two overlapping openreading frames (ORFs) encoding two separate proteins M2-1 and M2-2. The11 RSV proteins are: the RNA-binding nucleoprotein (N), thephosphoprotein (P), the large polymerase protein (L), the attachmentglycoprotein (G), the fusion protein (F), the small hydrophobic (SH)surface glycoprotein, the internal matrix protein (M), the twononstructural proteins NS1 and NS2, and the M2-1 and M2-2 proteins. TheRSV gene order is: 3′-NS1-NS2-N-P-M-SH-G-F-M2-L. Each gene is flanked byshort conserved transcription signals called the gene-start (GS) signal,present on the upstream end of each gene and involved in initiatingtranscription of the respective gene, and the gene-end (GE) signal,present at the downstream end of each gene and involved in directingsynthesis of a polyA tail followed by release of the mRNA.

The RSV F and G proteins are the only RSV proteins known to induce RSVneutralizing antibodies, and are the major protective antigens. The Fprotein generally is considered to be is a more effective neutralizationand protective antigen than the G protein. F also is relativelywell-conserved among RSV strains, whereas the G protein can besubstantially divergent. The divergence in G is a major factor insegregating RSV strains into two antigenic subgroups, A and B (˜53% and˜90% amino acid sequence identity between the two subgroups for G and F,respectively). The tools and methods of the present disclosure focus onRSV strain A2 of subgroup A, but can readily be applied to other strainsof either subgroup.

Vaccines and antiviral drugs against RSV are in pre-clinical andclinical development by a number of investigators; however, no vaccinesor antiviral drugs suitable for routine use against RSV are commerciallyavailable.

The development of RSV vaccines has been in progress since the 1960'sbut has been complicated by a number of factors. For example,immunization of RSV-naive infants with inactivated RSV has been shown toprime for enhanced disease upon subsequent natural RSV infection, andstudies in experimental animals indicate that disease enhancement alsois associated with purified RSV subunit vaccines. However, enhanced RSVdisease has not been observed in association with live or live-vectoredRSV vaccines, and this important observation has been confirmed in anumber of clinical studies (Wright, et al., 2007, Vaccine 25:7372-7378).Thus, inactivated and subunit vaccines are contraindicated for infantsand young children, whereas appropriately-attenuated live andlive-vectored vaccines are acceptable for use in this population, whichis the primary vaccine target population.

Another obstacle to immune protection is that RSV replicates and causesdisease in the superficial cells of the respiratory airway lumen, whereimmune protection has reduced effectiveness. Thus, immune control of RSVinfection is inefficient and often incomplete, and it is important foran RSV vaccine to be as immunogenic as possible. Another obstacle to RSVvaccines is that the magnitude of the protective immune response isroughly proportional to the extent of virus replication (and antigenproduction). Thus, the attenuation of RSV necessary to make a livevaccine typically is accompanied by a reduction in replication andantigen synthesis, and a concomitant reduction in immunogenicity, andtherefore it is essential to identify a level of replication that iswell tolerated yet satisfactorily immunogenic.

Another aspect of RSV vaccine development is that the virus does notreplicate efficiently in most experimental animals, such as rodents andmonkeys. Chimpanzees are more permissive but are no longer available forRSV research. Therefore, RSV vaccine development is heavily dependent onclinical studies even in early stages of development. Additionally, RSVgrows only to moderate titers in cell culture and is often present inlong filaments that are difficult to purify. Further, RSV can readilylose infectivity during handling.

Another obstacle is the difficulty in identifying and developingattenuating mutations. Appropriate mutations must be attenuating invivo, but should be minimally restrictive to replication in vitro, sincethis is essential for efficient vaccine manufacture. Yet anotherobstacle is genetic instability that is characteristic of RNA viruses,whereby attenuating mutations can revert to the wild-type (wt)assignment or to an alternative assignment that confers a non-attenuatedphenotype.

The combined approach of sequence design and synthetic biology allowsthe generation of DNA molecules with extensive targeted modifications.Synonymous genome recoding, in which one or more ORFs of a microbialpathogen are modified at the nucleotide level without affecting aminoacid coding, currently is being widely evaluated to reduce pathogenfitness and create potential live-attenuated vaccines, particularly forRNA viruses. The main strategies for attenuation by synonymous genomerecoding are: codon-deoptimization (CD), codon-pair-deoptimization(CPD), and increasing the dinucleotide CpG and UpA content (which isusually the result of CD and CPD).

Deoptimized virus genomes contain dozens to thousands of silentnucleotide mutations in one or more ORFs. Presumably, attenuation isbased on the sum of many individual mutations. This mutationmultiplicity is expected to confer stability against substantialde-attenuation, as the high number of mutations would present asignificant barrier against reversion to virulence. In principle, on thebackground of thousands of attenuating mutations, any single-sitereversion should yield only a minuscule selective advantage. The mostlikely path to reversion imaginable under this model is the progressiveaccumulation of many individual mutations, providing for a slowprogression of de-attenuation.

To date, genetic stability studies of large-scale deoptimized viruseshave shown that de-attenuation indeed appears to be low, suggesting thatthese viruses are genetically stable. However, an important limitationof these studies is that the de-optimized viruses generally have notbeen subjected to strong selective pressure that would favor theoutgrowth of viruses with de-attenuating mutations.

Thus, there continues to be a need for live attenuated RSV strains thatreplicate efficiently in vitro, and are maximally immunogenic,attenuated, and refractory to de-attenuation in vivo.

SUMMARY OF THE INVENTION

Disclosed herein are presumptively de-attenuating mutations in vitrothat are useful, either individually or in combination with other knownmutations, in producing recombinant strains of human respiratorysyncytial virus (RSV) exhibiting attenuation phenotypes in vivo. Furtherdisclosed herein are novel live-attenuated RSV strains suitable for useas RSV vaccines. Also provided herein are methods and compositionsrelated to the expression of the disclosed viruses. For example,isolated polynucleotide molecules that include a nucleic acid sequenceencoding the genome or antigenome of the described viruses aredisclosed.

In one embodiment, the present invention includes an isolatedpolynucleotide molecule encoding a recombinant respiratory syncytialvirus (RSV) variant having an attenuated phenotype comprising a RSVgenome or antigenome sequence, wherein the RSV genome or antigenome ismodified by a mutation in the L ORF at a position corresponding to T1166of the L protein in SEQ ID NO:11.

In some embodiments, the RSV genome or antigenome is further modified bya mutation selected from the group consisting of (i) a mutation in theM2-1 ORF at a position corresponding to N88 or A73 of the M2-1 proteinin SEQ ID NO:9; (ii) a mutation in the N ORF at a position correspondingto K136 of the N protein in SEQ ID NO:3; (iii) a mutation in the P ORFat a position corresponding to E114 of the P protein in SEQ ID NO:4; and(iv) combinations thereof. In some embodiments, the RSV genome orantigenome is further modified by a mutation selected from the groupconsisting of (i) a mutation in the M2-1 ORF at a position correspondingto N88 of the M2-1 protein in SEQ ID NO:9; (ii) a mutation in the N ORFat a position corresponding to K136 of the N protein in SEQ ID NO:3;(iii) a mutation in the P ORF at a position corresponding to E114 of theP protein in SEQ ID NO:4; and (iv) combinations thereof. In someembodiments, the RSV genome or antigenome is further modified by amutation selected from the group consisting of (i) a mutation in theM2-1 ORF at a position corresponding to A73 of the M2-1 protein in SEQID NO:9; (ii) a mutation in the N ORF at a position corresponding toK136 of the N protein in SEQ ID NO:3; (iii) a mutation in the P ORF at aposition corresponding to E114 of the P protein in SEQ ID NO:4; and (iv)combinations thereof. In some embodiments, the RSV genome or antigenomeis modified by at least two of mutations (i)-(iii). In some embodiments,the RSV genome or antigenome is modified by all of mutations (i)-(iii).

In some embodiments, the mutation in the L ORF at a positioncorresponding to T1166 of the L protein in SEQ ID NO:11 is T11661. Insome embodiments, (a) the mutation in the M2-1 ORF at a positioncorresponding to N88 of the M2-1 protein in SEQ ID NO:9 is N88K and themutation in the M2-1 ORF at a position corresponding to A73 of the M2-1protein in SEQ ID NO:9 is A73S; (b) the mutation in the N ORF at aposition corresponding to K136 of the N protein in SEQ ID NO:3 is K136R;and (c) the mutation in the P ORF at a position corresponding to E114 ofthe P protein in SEQ ID NO:4 is E114V. In some embodiments, the RSVgenome or antigenome is modified by at least two of mutations a-c. Insome embodiments, the RSV genome or antigenome is modified by all ofmutations a-c. In some embodiments, the mutation in the L ORF at aposition corresponding to T1166 of the L protein in SEQ ID NO:11 isT11661.

In some embodiments, the RSV genome or antigenome is modified by themutations corresponding to T11661 in the L protein in SEQ ID NO:11, N88Kin the M2-1 protein in SEQ ID NO:9, K136R in the N protein in SEQ IDNO:3 and E114V in the P protein in SEQ ID NO:4. In some embodiments, theRSV genome or antigenome is modified by the mutations corresponding toT11661 in the L protein in SEQ ID NO:11, A73S in the M2-1 protein in SEQID NO:9, K136R in the N protein in SEQ ID NO:3 and E114V in the Pprotein in SEQ ID NO:4.

In another embodiment, the present invention includes an isolatedpolynucleotide molecule encoding a recombinant respiratory syncytialvirus (RSV) variant having an attenuated phenotype comprising a RSVgenome or antigenome sequence, wherein the RSV genome or antigenome ismodified by one or more mutations selected from the positions recited inTable S1. In some embodiments, the RSV genome or antigenome is modifiedby one or more mutations selected from the positions recited in TableS1-A. In some embodiments, the RSV genome or antigenome is modified byone or more mutations selected from the positions recited in Table S1-B.

In another embodiment, the present invention includes an isolatedpolynucleotide molecule encoding a recombinant respiratory syncytialvirus (RSV) variant having an attenuated phenotype comprising a RSVgenome or antigenome sequence, wherein the RSV genome or antigenome ismodified by one or more mutations selected from the positions recited inTable S2. In some embodiments, the RSV genome or antigenome is modifiedby one or more mutations selected from the positions recited in TableS2-A. In some embodiments, the RSV genome or antigenome is modified byone or more mutations selected from the positions recited in Table S2-B.

In another embodiment, the present invention includes an isolatedpolynucleotide molecule encoding a recombinant respiratory syncytialvirus (RSV) variant having an attenuated phenotype comprising a RSVgenome or antigenome sequence, wherein the RSV genome or antigenome ismodified by one or more mutations selected from the positions recited inTable S3. In some embodiments, the RSV genome or antigenome is modifiedby one or more mutations selected from the positions recited in TableS3-A. In some embodiments, the RSV genome or antigenome is modified byone or more mutations selected from the positions recited in Table S3-B.

In some embodiments, the RSV genome or antigenome comprises a deletionin at least one of the proteins selected from M2-2, NS1 and NS2. In someembodiments, the RSV genome or antigenome is codon-pair deoptimized. Insome embodiments, the L-ORF of the RSV genome or antigenome iscodon-pair deoptimized.

In some embodiments, the present invention includes a polynucleotidemolecule comprising a nucleotide sequence that is at least about 80%identical to the nucleotide sequence of SEQ ID NO:14. In someembodiments, the present invention includes a polynucleotide moleculecomprising nucleotide sequence that is at least about 90% identical tothe nucleotide sequence of SEQ ID NO:14. In some embodiments, thepresent invention includes a polynucleotide molecule comprisingnucleotide sequence that is at least about 95% identical to thenucleotide sequence of SEQ ID NO:14. In some embodiments, the presentinvention includes a polynucleotide molecule comprising the nucleotidesequence of SEQ ID NO:14.

In some embodiments, the present invention includes a vector comprisingthe isolated polynucleotide molecules described above. In someembodiments, the present invention includes a cell comprising theisolated polynucleotide molecules described above.

In some embodiments, the present invention includes a pharmaceuticalcomposition comprising an immunologically effective amount of therecombinant RSV variant encoded by the isolated polynucleotide moleculesdescribed above. In some embodiments, the present invention includes amethod of vaccinating a subject against RSV comprising administering thepharmaceutical composition. In some embodiments, the present inventionincludes a method of inducing an immune response comprisingadministering the pharmaceutical composition. In some embodiments, thepharmaceutical composition is administered intranasally. In someembodiments, the pharmaceutical composition is administered viainjection, aerosol delivery, nasal spray or nasal droplets.

In some embodiments, the present invention includes a live attenuatedRSV vaccine comprising the recombinant RSV variant encoded by theisolated polynucleotides described above. In some embodiments, thepresent invention includes a pharmaceutical composition comprising theRSV vaccine. In some embodiments, the present invention includes amethod of making the vaccine comprising expressing the isolatedpolynucleotide molecules described above.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that Min_FLC was phenotypically stable during a temperaturestress test, but Min_L was not. (A) Gene maps of Min_L and Min_FLCshowing ORFs that are wt (grey) or CPD (black). The number of introducedmutations in each virus and the shut-off temperature (T_(SH)) in Verocells are indicated. (B-E) Incubation temperature and virus yield ateach passage level during serial passage in temperature stress tests.Replicate cultures of Vero cells in T25 flasks were infected with theindicated virus at MOI 0.1 and, when the viral cytopathic effect wasextensive, or when cells started to detach (for passages of Min_FLC at37° C. and beyond, (C)), flasks were harvested and clarified culturefluids were passaged 1:5 to a fresh flask. Each starting replicate flaskinitiated an independent serial passage (lineage). Aliquots of clarifiedculture fluids were frozen for titration and sequence analysis. (B, C)Temperature stress test of Min_FLC. Two control flasks inoculated withMin_FLC (B) were passaged 18 times at the permissive temperature of 32°C. Ten additional replicates (C) were passaged from 32 to 40° C. with 2passages at each temperature. (D, E) Temperature stress test of Min_L.Two control flasks inoculated with Min_L (D) were passaged 8 times atthe permissive temperature of 32° C. Ten additional replicates (E) werepassaged from 37 to 40° C. with 2 passages at each temperature. Lineages#3 and 8 are shown. (F, G) Accumulation of most abundant mutations (>30%of the reads in at least one passage) in lineages #3 (F) and #8 (G)during the passage series, determined by deep sequencing (see Tables S2and S3 for detailed data).

FIG. 2 shows that M2-1 mutations [A73S] and [N88K] segregated intodifferent viral subpopulations. (A) Percentage of deep sequencing readsthat contained M2-1 mutation [A73S] or [N88K] at P6 (the second passageat 39° C.) of each of the 10 lineages from the experiment in FIG. 1,panel (E). (B) Lack of linkage between M2-1 mutations [A73S] and [N88K],illustrated by the percentage of deep sequencing reads that containedthe indicated combinations of assignments at codons 73 (wt versus[A73S]) and 88 (wt versus [N88K]) in the same read; based on reads fromthe experiment in FIG. 1, panel (F) that spanned both codons. (C) Extentof linkage between M2-1 mutations [A73S] and [N88K] and other mutationsduring the first 4 passages of lineage #3 (FIG. 1, panels (E) and (F)),determined by PacBio sequencing of continuous reads corresponding to an8.2 kb region of the RSV genome from the 3′ end to the middle of theM2-2 ORF. Four major virus subpopulations were identified, and mutationsthat are linked on the same genomes are indicated.

FIG. 3 shows the effects of specific mutations on the temperaturesensitivity and in vitro replication of Min_L derivatives. Five majormutations identified in lineage #3 (N[K136R], P[E114V], L[T1166I],M2-1[N88K] and M2-1[A73S], FIG. 1, panel (F)) were introducedindividually and in combinations by site-directed mutagenesis andreverse genetics into Min_L for phenotypic analysis. The Min_L-derivedviruses were named based on the gene names bearing the introducedmutations, with the M2-1 mutation specified in brackets. (A) Mutationsare indicated in the viral genome map. (B) T_(SH), determined by theefficiency of plaque formation at 32, 35, 36, 37, 38, 39, and 40° C.using published methods. The experiment was done 4 times for viruses #1,5, 12 and 14, 3 times for viruses #2, 3, 7, 8 and 9, 2 times for viruses#4 and 11 and once for viruses #6, 10 and 13 (bars graphs: medians andrange). (C, D) Replication of Min_L-derived mutants in vitro. Vero cellswere infected at 32° C. and 37° C. (MOI of 0.01). Titers correspond tothe mean of two replicate titrations of two replicates for each timepoint. The standard deviation is indicated. Due to the large number ofviruses, the analysis was divided between experiments #1 (C) and #2 (D).

FIG. 4 shows the effects of specific mutations on RNA synthesis andplaque size of Min_L derivatives. (A-E) Replicate cultures of Vero cellswere infected (MOI of 3) with the indicated viruses. Cultures wereharvested every 4 h from 4 to 24 hpi for analysis of cell-associatedRNA, protein, and virus. (A) Positive-sense viral RNA (i.e.,mRNA+antigenome) was quantified in triplicate by strand-specificRT-qPCR. Data for P are shown. QPCR results were analyzed using thecomparative threshold cycle (ΔCt) method, normalized to 18S rRNA, andexpressed as loge fold increase over the Min_L 4 h time point. (B)Quantification of P protein expression by Western blotting. (C)Quantification of L mRNA +antigenome by strand-specific RT-qPCR (foldincrease relative to the 4 hpi time point, calculated separately foreach virus, as different primer-probes sets were required for wt L genein wt rRSV versus the CPD L gene present in Min_L and its derivatives).For wt L and CPD L, data were derived from 3 and 4 differentprimer-probe sets, respectively, designed along the L ORFs, and themedian values with ranges are shown. (D) Quantification ofcell-associated genomic RNA by strand-specific RT-qPCR, expressed asfold increase over the 4 hpi time point of Min_L. (E) Virus titers fromcultures incubated at 32 and 37° C., assayed at 32° C. (F-G) Virusplaque sizes. Vero cells were infected with 30 pfu per 2 cm² well of wtrRSV, Min_L, and Min_L-derived mutants and incubated undermethylcellulose at 32° C. for 12 days. Plaques were visualized byimmunostaining and quantified by IR imaging (Licor) using Image J. (F)Representative pictures of virus plaque sizes. (G) Plaques sizedistribution of the indicated viruses. A minimum of 1000 plaques pervirus was measured (*=p≤0.05).

FIG. 5 show the analysis of Min_L derivatives in rodents, whichindicates differing effects of M2-1 mutations A73S and N88K andidentifies the improved vaccine candidate NPM2-1[N88K]L. Replication ofMin_L, Min_L mutants and wt rRSV in mice at day 4 (A) and 5 pi (B) or inhamsters at day 3 pi (C). Groups of 20 mice (A-B) or 18 hamsters (C)were infected intranasally with 10⁶ pfu of the indicated virus/animal.At day 4 (A), 5 (B) and 10 pi (data not shown) for the mouse study or atday 3 for the hamster study (C), RSV titers in nasal turbinates (NT) andlungs were determined as described in the experimental proceduressection. The limit of detection is indicated by a dotted line. (D)RSV-neutralizing antibodies at day 26 in hamsters from 9 hamsters pergroup. The 60% plaque reduction neutralizing antibody titers (PRNT₆₀)were determined as described previously. Statistical differencescompared with wt rRSV indicated on the top of each graph; statisticaldifferences between Min_L and the Min_L-derived mutants indicated bybrackets (*p≤0.05, **p≤0.01, ***p≤0.001 and ****p≤0.0001).

FIG. 6 shows molecular modeling of the impact of de-attenuatingmutations on the M2-1 tetramer. (A) Top view of wt M2-1 tetramer. (B)Enlargement of one wt tetramer's region that contains amino acids A73and N88. (C) Molecular dynamics snapshot of the region proximal to theS73 mutation. The [A73S] mutation is shown and the arrows indicate thepredicted new hydrogen bond. (D) Molecular dynamics snapshot of the K88mutant region. The [N88K] mutation is indicated and an arrow indicatesthe expected new salt-bridge.

FIG. 7 shows the minimal accumulation of adventitious mutations inMin_FLC during 18 passages at 32° C. Min_FLC was subjected to 18passages at 32° C. At the end of passage 18, viral RNA was extractedfrom lineage #1 (A) and #2 (B), and the complete genome was amplified byoverlapping RT-PCR and analyzed by deep sequencing (Ion Torrent).Adventitious mutations (which are not specifically identified) areindicated by bars showing their genome position and relative abundance.WT genes are colored in grey shading, while CPD genes are colored inblack shading.

FIG. 8 shows the minimal accumulation of adventitious mutations in Min_Lduring 6 passages at 32° C. Min_L was sequentially passed 8 times at 32°C. on Vero cells. At the end of passage 6, viral RNA was extracted fromlineage #1 (A) and #2 (B), and the complete genome was amplified byoverlapping RT-PCR and analyzed by deep sequencing (Ion Torrent).Adventitious mutations are indicated by bars showing their genomeposition and relative abundance; the specific nucleotide changes are notindicated. WT genes are colored in grey shading, while CPD genes arecolored in black shading.

FIG. 9 shows the contributions of specific mutations to the phenotypesof Min_L derivatives: RT-qPCR of cell-associated positive-sense RNA(mRNA+antigenome). The RT-qPCR data during infection of Vero cells withwt rRSV, Min_L, and Min_L-derivatives for the NS1, NS2, N, P, M, SH, G,F, and M2 mRNAs are shown here.

FIG. 10 shows the contributions of specific mutations to the phenotypesof Min_L derivatives: protein expression of Min_L and Min_L-derivedmutants. Vero cells were infected at an MOI of 3 pfu/cell at 32 or 37°C. with Min_L, M2-1[A73S], M2-1[N88K], PM2-1[N88K], NPM2-1[N88K]L or wtrRSV. Every 4 h from 4 to 24 hpi, total cell lysates were harvested fromone well of a 6-well plate in NuPage LDS sample buffer (LifeTechnologies). Western blot analysis of NS1, NS2, N, P, G, F and M2-1,was performed as described in the materials and methods section. TheGAPDH protein was used as a loading control. Membranes were scanned onthe Odyssey® Infrared Imaging System. Data collected was analyzed usingOdyssey software, version 3.0. For quantification of identified RSVproteins of interest, background fluorescence was corrected. Valuesreported indicate the median fluorescence intensity per protein band.

FIG. 11 shows the T_(S)H of Min_FLC derived mutants. The effects of themutations involved in the loss of temperature sensitivity of Min_L onMin_FLC temperature sensitivity were investigated. (A) To do so,mutations that were identified in Min_L lineage #3 (in N, P, M2-1 [N88K]and L genes) or #8 (M2-1 mutation [A73S]) were re-introduced alone or inthe indicated combinations into Min_FLC backbone and the derived cDNAwas completely sequenced by Sanger sequencing. Viruses were rescued byreverse genetics, passaged once, and virus stocks at P2 were titrated.Because of the low virus titer of most of the virus stocks, only themutant virus Min_FLC M2-1[A735] was completely sequenced by Sangersequencing. (B) The is phenotype of some of these Min_FLC-derivedmutants was evaluated by efficiency of plaque formation at 32, 35, 36,37, 38, 39, and 40° C. Plaque assays were performed on Vero cells induplicate, and incubated in sealed caskets at various temperatures intemperature controlled water-baths as previously described. Theexperiment was done twice. The median values and the standard deviationis indicated.

FIG. 12 shows that NPM2-1[N88K]L is phenotypically stable under atemperature stress test. (A) Schematic representation of the RSV genomeorganization. The abbreviated gene name is indicated. Genes with wt orCPD ORFs are indicted by grey and black shading, respectively. Mutationsin N, P, M2-1 and L that were identified in lineage #3 and introducedinto Min_L backbone to generate the NPM2-1[N88K]L virus are indicated bybars in the virus genome. (B) Final virus titers at 32° C. and (C) fromthe temperature stress passages (increasing temperatures are indicatedbelow the x axis). Each symbol represents one replicate.

FIG. 13 shows the Amino acid sequences of the RSV proteins NS1, NS2, N,P, M, SH, G, F, M2-1, M2-2 and L. These are represented by SEQ IDNO:1-11 respectively.

FIG. 14 shows Nucleotide sequence of recombinant RSVMin_L-NPM2-1[N88K]L. This is represented by SEQ ID NO:14.

DETAILED DESCRIPTION

Provided herein are recombinant RSV strains suitable for use asattenuated, live vaccines in humans. The RSV strains may be produced byintroducing one or more mutations in the RSV genome or antigenomesequence selected from the positions described below and listed intables S1, S2 and S3. These mutations were identified by evaluatingphenotypic reversion of de-optimized human respiratory syncytial virus(RSV) vaccine candidates in the context of strong selective pressure.

Codon-pair de-optimized (CPD) versions of RSV were attenuated andtemperature-sensitive. During serial passage at progressively increasingtemperature, a CPD RSV containing 2,692 synonymous mutations in 9 of 11ORFs, named Min_FLC, did not lose temperature sensitivity and remainedgenetically and phenotypically stable during 7 months of passage invitro at the permissive temperature of 32° C., as well as underconditions of increasing temperature during passage. This is strongevidence for the stability of Min_FLC, and validates the safety of CPDof multiple genes for the development of live-attenuated vaccines forRSV and related viruses, provided that extensive CPD is employed.

However, a CPD RSV in which only the polymerase L ORF was deoptimized,named Min_L, was highly stable at 32° C. but surprisingly, despite thelarge number of changes involved in its CPD, quickly lost substantialattenuation and evolved to escape temperature sensitivity restriction.Comprehensive sequence analysis of virus populations identified manydifferent potentially de-attenuating mutations in the L ORF,surprisingly many appearing in other ORFs that had not been subjected toCPD. In particular. deep sequencing of the Min_L lineages identifiedmutations in all but the NS2 ORF, rather than specifically in the CPD LORF, as might have been expected. Surprisingly, many of the mutations inL occurred at nucleotides and codons that were not involved in CPD.These are shown in tables S1, S2 and S3.

Some of these presumptive de-attenuating mutations, while beingde-attenuating in vitro, when incorporated into Min_L with otherpresumptive de-attenuating mutations, were found to have the surprisingeffect of being further attenuating than Min_L in vivo.

In one exemplary embodiment, Min_L-NPM2-1[N88K]L (also referred hereinas NPM2-1[N88K]L) described in detail below (nucleotide sequence shownin FIG. 14), was more attenuated than Min_L in vivo rather than beingde-attenuated. Furthermore, while the NPM2-1[N88K]L virus was highlyattenuated in vivo, surprisingly it was as immunogenic as wild type RSV.Additionally, it did not acquire any significant mutations during afurther stress test (see FIG. 12), and thus was more genetically stablethan Min_L. Thus, Min_L-NPM2-1[N88K]L represented a substantialimprovement over Min_L as a vaccine candidate for the following reasons.It was significantly more attenuated in vivo than Min_L, yet asimmunogenic as wt RSV. It did not accumulate additional mutations whenpassaged in stress tests at 39-40° C. It exhibited increased replicationcompared to Min_L in Vero cells, which is important for vaccinemanufacture. Furthermore, as described in detail below, since theM2-1[N88K] and [A73S] mutations are incompatible, this virus is highlyrefractory to acquiring the M2-1[A735] mutation that was de-attenuatingin the hamster model.

Accordingly, provided herein are recombinant RSV strains having anattenuated phenotype comprising a RSV genome or antigenome sequence,wherein the RSV genome or antigenome is modified by one or moremutations selected from Table S1, S2 or S3. The mutations listed inTables S1, S2 or S3 are presumptive de-attenuating mutations butsurprisingly may impart attenuation phenotype in vivo. Mutations listedin Tables S1, S2 and S3 present in ≥25% reads are listed in Tables S1-A,S2-A and S3-A respectively, and the most abundant mutations present in≥50% reads are listed in Tables S1-B, S2-B and S3-B, respectively.

In one embodiment, the invention comprises an isolated polynucleotidemolecule encoding a recombinant respiratory syncytial virus (RSV)variant having an attenuated phenotype comprising a RSV genome orantigenome sequence, wherein the RSV genome or antigenome is modified byone or more mutations selected from the positions recited in Table S1.In some embodiments, the RSV genome or antigenome is modified by one ormore mutations selected from the positions recited in Table S1-A. Insome embodiments, the RSV genome or antigenome is modified by one ormore mutations selected from the positions recited in Table S1-B.

In one embodiment, the invention comprises an isolated polynucleotidemolecule encoding a recombinant respiratory syncytial virus (RSV)variant having an attenuated phenotype comprising a RSV genome orantigenome sequence, wherein the RSV genome or antigenome is modified byone or more mutations selected from the positions recited in Table S2.In some embodiments, the RSV genome or antigenome is modified by one ormore mutations selected from the positions recited in Table S2-A. Insome embodiments, the RSV genome or antigenome is modified by one ormore mutations selected from the positions recited in Table S2-B.

In one embodiment, the invention comprises an isolated polynucleotidemolecule encoding a recombinant respiratory syncytial virus (RSV)variant having an attenuated phenotype comprising a RSV genome orantigenome sequence, wherein the RSV genome or antigenome is modified byone or more mutations selected from the positions recited in Table S3.In some embodiments, the RSV genome or antigenome is modified by one ormore mutations selected from the positions recited in Table S3-A. Insome embodiments, the RSV genome or antigenome is modified by one ormore mutations selected from the positions recited in Table S3-B.

In some embodiments, the RSV genome or antigenome may be modified by amutation in the L ORF at a position corresponding to or in the codonencoding amino acid residue 1166 of the L protein. In some embodiments,the mutation in the L ORF may be at a position corresponding to T1166 ofthe L protein as shown in the sequence of FIG. 13 (SEQ ID NO:11). Insome embodiments, the mutation may cause an amino acid other thanThreonine to be encoded at that position. In some embodiments, themutation may cause isoleucine to be encoded at that position. Thismutation, T11661 is listed in Tables S1, S1-A and S1-B.

In some embodiments, the RSV genome or antigenome may be furthermodified by one or more additional mutations. The additional mutationsmay be in the L ORF or any of the other ORFs. For example, in someembodiments, the additional one or more mutations may be in M2-1 ORF,the N ORF or the P ORF.

In some embodiments, the additional mutation may be in the M2-1 ORF at aposition corresponding to or in the codon encoding amino acid residue 88or 73 of the M2-1 protein. In some embodiments, the mutation in the M2-1ORF may be at a position corresponding to N88 or A73 of the M2-1 proteinas shown in sequence of FIG. 13 (SEQ ID NO:9). In some embodiments, theadditional mutation in the M2-1 ORF may be at a position correspondingto position N88 of the M2-1 protein, and may cause an amino acid otherthan asparagine to be encoded at that position. In some embodiments, itmay cause lysine to be encoded at that position (N88K). In someembodiments, the additional mutation in the M2-1 ORF may be at aposition corresponding to position A73 of the M2-1 protein, and maycause an amino acid other than alanine to be encoded at that position.In some embodiments, the mutation in the codon encoding amino acidresidue 73 of the M2-1 protein may cause serine to be encoded at thatposition (A73S).

In some embodiments, the additional mutation may be in the N ORF at aposition corresponding to or in the codon encoding amino acid residue136 of the N protein. In some embodiments, the mutation in the N ORF maybe at a position corresponding to K136 of the N protein as shown insequence of FIG. 13 (SEQ ID NO:3), and may cause an amino acid otherthan lysine to be encoded at that position. In some embodiments, themutation in the codon encoding amino acid residue 136 of the N proteinmay cause arginine to be encoded at that position (K136R).

In some embodiments, the additional mutation may be in the P ORF at thecodon encoding amino acid residue 114 of the P protein. In someembodiments, the mutation in the P ORF may be at a positioncorresponding to E114 of the P protein as shown in sequence of FIG. 13(SEQ ID NO:4), and may cause an amino acid other than glutamic acid tobe encoded at that position. In some embodiments, the mutation in thecodon encoding amino acid residue 136 of the P protein may cause valineto be encoded at that position (E114V).

In some embodiments, the RSV genome or antigenome may be modified tocomprise at least two of the mutations described above. For example, itmay comprise at least two mutations at positions corresponding to N88 orA73 in M2-1 protein, K136 in the N protein, E114 in the P protein andT1166 in the L protein. In some embodiments, the RSV genome orantigenome may be modified to comprise all four of the mutationsdescribed above. Thus, for example, in some embodiments it may comprisemutations at positions corresponding to N88 in M2-1 protein, K136 in theN protein, E114 in the P protein and T1166 in the L protein. In someembodiments it may comprise mutations at positions corresponding to A73in M2-1 protein, K136 in the N protein, E114 in the P protein and T1166in the L protein.

In some embodiments, the isolated polynucleotide molecule may comprise aRSV genome or antigenome modified by mutations corresponding to orencoding N88K in M2-1 protein, K136R in the N protein, E114V in the Pprotein and T11661 in the L protein. In some embodiments, the isolatedpolynucleotide molecule may comprise a RSV genome or antigenome modifiedby mutations corresponding to or encoding A73S in M2-1 protein, K136R inthe N protein, E114V in the P protein and T11661 in the L protein.

In some embodiments, the RSV genome or antigenome may be deoptimized.Thus, in some embodiments, the attenuated RSVs described herein areproduced by introducing codon changes in the viral genome that are notoptimally processed by the host cell. The majority of these mutations donot cause a change in the resulting amino acid of proteins encoded bythe viral genome, thus allowing for the production of viruses that havethe same antigenic features of wild-type viruses. It should beunderstood, however, that widespread noncoding changes to the codons ofthe viral genome may result in a selective pressure that gives rise toone or more amino acid mutations in the viruses described herein.

This substitution of synonymous codons alters various parameters,including codon bias, codon pair bias, density of deoptimized codons anddeoptimized codon pairs, RNA secondary structure, CpG dinucleotidecontent, C+G content, translation frameshift sites, translation pausesites, the presence or absence of tissue specific micro RNA recognitionsequences, or any combination thereof, in the genome. The mainstrategies for attenuation by synonymous genome recoding are:codon-deoptimization (CD), codon-pair-deoptimization (CPD), andincreasing the dinucleotide CpG and UpA content (which is usually theresult of CD and CPD). In some embodiments, any one of the ORFs of theRSV, including NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2 and L, may becodon-pair deoptimized. In some embodiments, any two or more of the ORFsof the RSV may be codon-pair deoptimized. In some embodiments, any threeor more of the ORFs of the RSV may be codon-pair deoptimized.

In some embodiments, any four or more of the ORFs of the RSV may becodon-pair deoptimized. In some embodiments, any five or more of theORFs of the RSV may be codon-pair deoptimized. In some embodiments, anysix or more of the ORFs of the RSV may be codon-pair deoptimized. Insome embodiments, any seven or more of the ORFs of the RSV may becodon-pair deoptimized. In some embodiments, any eight or more of theORFs of the RSV may be codon-pair deoptimized. In some embodiments, anynine or more of the ORFs of the RSV may be codon-pair deoptimized. Insome embodiments, any ten or more of the ORFs of the RSV may becodon-pair deoptimized. In some embodiments, all of the ORFs of the RSVmay be codon-pair deoptimized. In some embodiments, the L ORF of the RSVmay be codon-pair deoptimized. In some embodiments, the NS1, NS2, N, P,M and SH ORFs of the RSV may be codon-pair deoptimized. In someembodiments, the G and F ORFs of the RSV may be codon-pair deoptimized.In some embodiments, the NS1, NS2, N, P, M, SH, G, F and L ORFs of theRSV may be codon-pair deoptimized.

In some embodiments, the isolated polynucleotide molecule may comprise anucleotide sequence that is at least about at least about 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83 , 84, 85, 86, 87, 88, 89, 90,91, 92, 93, 94, 95, 96, 97, 98, 99 or 100 percent identity (or anypercent identity in between) to the nucleotide sequence of SEQ ID NO:14.In some embodiments, the isolated polynucleotide molecule may comprise anucleotide sequence that is at least about 80% identical to thenucleotide sequence of SEQ ID NO:14. In some embodiments, the isolatedpolynucleotide molecule may comprise a nucleotide sequence that is atleast about 90% identical to the nucleotide sequence of SEQ ID NO:14. Insome embodiments, the isolated polynucleotide molecule may comprise anucleotide sequence that is at least about 95% identical to thenucleotide sequence of SEQ ID NO:14. In some embodiments, the isolatedpolynucleotide molecule may comprise an isolated polynucleotidecomprising the nucleotide sequence of SEQ ID NO:14.

In some embodiments, the described viruses may be combined with knownattenuating mutations of RSV and of related viruses to yield gradedattenuation phenotypes. A number of such mutations are known in the artand are encompassed in this invention. For example, in some embodiments,the RSV genome or antigenome may be modified by a deletion in the M2-2ORF, the NS1 ORF or the NS2 ORF.

Given that a variety of RSV strains exist (e.g., RSV A2, RSV B1, RSVLong), those skilled in the art will appreciate that certain strains ofRSV may have nucleotide or amino acid insertions or deletions that alterthe position of a given residue. For example, if a protein of anotherRSV strain had, in comparison with strain A2, two additional amino acidsin the upstream end of the protein, this would cause the amino acidnumbering of downstream residues relative to strain A2 to increase by anincrement of two. However, because these strains share a large degree ofsequence identity, those skilled in the art would be able to determinethe location of corresponding sequences by simply aligning thenucleotide or amino acid sequence of the A2 reference strain with thatof the strain in question. Therefore, it should be understood that theamino acid and nucleotide positions described herein, thoughspecifically enumerated in the context of this disclosure, cancorrespond to other positions when a sequence shift has occurred or dueto sequence variation between virus strains. In the comparison of aprotein, or protein segment, or gene, or genome, or genome segmentbetween two or more related viruses, a “corresponding” amino acid ornucleotide residue is one that is thought to be exactly or approximatelyequivalent in function in the different species.

The numbering used in this disclosure is based on the amino acidsequence of the wild-type RSV A2 strain (GenBank accession numberM74568, which is expressly incorporated herein) and all nucleotidesequences described are in positive-sense. The amino acid sequences ofthe 11 RSV proteins NS1, NS2, N, P, M, SH, G, F, M2-1, M2-2, and L areshown in FIG. 13 and represented in SEQ ID NOS: 1, 2, 3, 4, 5, 6, 7, 8,9, 10 and 11 respectively.

In some embodiments of the present invention, the recombinant RSVstrains may be derived from the recombinant version of strain A2 that iscalled D46. The complete sequence of D46 is shown in U.S. Pat. No.6,790,449 (GenBank accession number KT992094, which is expresslyincorporated herein). (In some instances and publications, the parentvirus and sequence is called D53 rather than D46, a book-keepingdifference that refers to the strain of bacteria used to propagate theantigenomic cDNA and has no other known significance or effect. For thepurposes of this invention, D46 and D53 are interchangeable.) Thenucleotide sequence of D46 differs from the sequence of RSV A2 strainM74568 in 25 nucleotide positions, which includes a 1-nt insert atposition 1099.

Additional mutations may be further introduced in combination with themutations defined above to construct additional viral strains withdesired characteristics. For example, the added mutations may specifydifferent magnitudes of attenuation, and thus give incremental increasesin attenuation. Thus, candidate vaccine strains can be furtherattenuated by incorporation of at least one, and preferably two or moredifferent attenuating mutations, for example mutations identified from apanel of known, biologically derived mutant RSV strains. A number ofsuch mutations are discussed here as examples. From this exemplary panela large “menu” of attenuating mutations can be created, in which eachmutation can be combined with any other mutation(s) within the panel forcalibrating the level of attenuation and other desirable phenotypes.Additional attenuating mutations may be identified in non-RSV negativestranded RNA viruses and incorporated in RSV mutants of the invention bymapping the mutation to a corresponding, homologous site in therecipient RSV genome or antigenome and mutating the existing sequence inthe recipient to the mutant genotype (either by an identical orconservative mutation). Additional useful mutations can be determinedempirically by mutational analysis using recombinant minigenome systemsand infectious virus as described in the references incorporated herein.

The recombinant RSV vaccine strains of the present invention were madeusing a recombinant DNA-based technique called reverse genetics(Collins, et al. 1995. Proc Natl Acad Sci USA 92:11563-11567). Thissystem allows de novo recovery of infectious virus entirely from cDNA ina qualified cell substrate under defined conditions. Reverse geneticsprovides a means to introduce predetermined mutations into the RSVgenome via the cDNA intermediate. Specific attenuating mutations werecharacterized in preclinical studies and combined to achieve the desiredlevel of attenuation. Derivation of vaccine viruses from cDNA minimizesthe risk of contamination with adventitious agents and helps to keep thepassage history brief and well documented. Once recovered, theengineered virus strains propagate in the same manner as a biologicallyderived virus. As a result of passage and amplification, the vaccineviruses do not contain recombinant DNA from the original recovery.

The recombinant virus strains that contain various combinations ofmutations discussed herein are for exemplary purposes only and are notmeant to limit the scope of the present invention. For example, in someembodiments, the recombinant RSV strains of the present inventionfurther comprise a deletion of the non-translated sequences. In oneembodiment, such deletion occurs in the downstream end of the SH gene,resulting in a mutation called the “6120 Mutation” herein. It involvesdeletion of 112 nucleotides of the downstream non-translated region ofthe SH gene and the introduction of five translationally-silent pointmutations in the last three codons and the termination codon of the SHgene (Bukreyev, et al. 2001. J Virol 75:12128-12140). Presence of theterm “LID” or “6120” in a recombinant virus name indicates that therecombinant virus contains the 6120 mutation.

The 6120 mutation stabilizes the antigenomic cDNA in bacteria so that itcould be more easily manipulated and prepared. In wt RSV, this mutationwas previously found to confer a 5-fold increase in replicationefficiency in vitro (Bukreyev, et al. 2001. J Virol 75:12128-12140),whereas it was not thought to increase replication efficiency in vivo.

The 6120 mutation was associated with increased replication inseronegative infants and children. Thus, the 6120 mutation providedanother means to shift the level of attenuation. Also, the deletion ofsequence exemplified by the 6120 mutation in the downstreamnon-translated region of the SH gene, but in principle could involve anycomparable genome sequence that does not contain a critical cis-actingsignal (Collins and Karron. 2013. Fields Virology 6th Edition, pp1086-1123). Genome regions that are candidates for deletion include, butare not limited to, non-translated regions in other genes, in theintergenic regions, and in the trailer region.

In some embodiments the recombinant RSV strains may comprise the “cp”mutation. This mutation refers to a set of five amino acid substitutionsin three proteins (N (V267I), F (E218A and T523I), and L (C319Y andH1690Y)) that together (on their own) confer an approximate 10-foldreduction in replication in seronegative chimpanzees, and a reduction inillness (Whitehead, et al. 1998. J Virol 72:4467-4471). We previouslyshowed that the cp mutation is associated with a moderate attenuationphenotype (Whitehead, et al. 1999. J Virol 72:4467-4471).

In addition, previous analysis of 6 biological viruses that had beenderived by chemical mutagenesis of cpRSV and selected for thetemperature-sensitive (ts) phenotype yielded a total of 6 independentmutations that each conferred a ts attenuation phenotype and could beused in various combinations. Five of these were amino acidsubstitutions in the L protein, which were named based on virus numberrather than sequence position: “955” (N43I), “530” (F521L), “248”(Q831L), “1009” (M1169V), and “1030” (Y1321N) (Juhasz, et al. 1999.Vaccine 17:1416-1424; Collins, et al. 1999. Adv Virus Res 54:423-451;Firestone, et al. 1996. Virology 225:419-422; Whitehead, et al. 1999. JVirol 73:871-877). The sixth mutation (called “404”) was a singlenucleotide change in the gene-start transcription signal of the M2 gene(GGGGCAAATA to GGGGCAAACA, mRNA-sense) (Whitehead, et al. 1998. Virology247:232-239). We recently used reverse genetics to increase the geneticstability of the 248 and 1030 mutations (Luongo, et al. 2009. Vaccine27:5667-5676; Luongo, et al. 2012. J Virol 86:10792-10804). In addition,we created a new attenuating mutation by deleting codon 1313 in the Lprotein and combining it with an I1314L substitution to confer increasedgenetic stability (Luongo, et al. 2013. J Virol 87:1985-1996).

In some embodiments, the recombinant strains may comprise one or morechanges in the F protein, e.g. the “HEK” mutation, which comprises twoamino acid substitutions in the F protein namely K66E and Q101P(described in Connors, et al. 1995. Virology 208:478-484; Whitehead, etal. 1998. J Virol 72:4467-4471). The introduction of the HEK amino acidassignments into the strain A2 F sequence of this disclosure results inan F protein amino acid sequence that is identical to that of anearly-passage (human embryonic kidney cell passage 7, HEK-7) of theoriginal clinical isolate of strain A2 (Connors, et al. 1995. Virology208:478-484; Whitehead, et al. 1998. J Virol 72:4467-4471). It resultsin an F protein that is much less fusogenic and is thought to representthe phenotype of the original A2 strain clinical isolate (Liang et al. JVirol 2015 89:9499-9510). The HEK F protein also forms a more stabletrimer (Liang et al. J Virol 2015 89:9499-9510). This may provide a moreauthentic and immunogenic form of the RSV F protein, possibly enrichedfor the highly immunogenic pre-fusion conformation (McLellan et al.Science 2013 340(6136):1113-7; Science 2013 342(6158):592-8.). Thus,mutations can be introduced with effects additional to effects on themagnitude of virus replication.

In some embodiments the recombinant strains may comprise one or morechanges in the L protein, e.g. the stabilized 1030 or the “1030s”mutation which comprises 1321K(AAA)/S1313(TCA) (Luongo, et al. 2012. JVirol 86:10792-10804).

In some embodiments the recombinant strains may comprise one or morechanges in the N protein, e.g. an amino substitution such as T24A.Deletion of the SH, NS1, and NS2 genes individually and in combinationhas been shown to yield viruses that retain their ability to replicatein cell culture but are attenuated in vivo in the following order ofincreasing magnitude: SH<NS2<NS1 (Bukreyev, et al. 1997. J Virol71:8973-8982; Whitehead, et al. 1999. J Virol 73:3438-3442; Teng, et al.2000. J Virol 74:9317-9321). Therefore, deletion or other mutations ofthe SH, NS2, or NS1 genes, or parts of their ORFs, may be combined witha mutation described here. For example, in some embodiments, therecombinant strains may comprise one or more changes in the SH protein,including an ablation or elimination of the SH protein. In someembodiments, the viral strains comprise a deletion in the SH gene. Forexample, in some embodiments, the viral strains comprise a 419nucleotide deletion at position 4197-4615 (4198-4616 of), denoted hereinas the “ASH” mutation. This deletion results in the deletion of Mgene-end, M/SH intergenic region, and deletion of the SH ORF as shown inFIG. 6. In some embodiments, the recombinant strains may comprise one ormore changes in the NS1 or the NS2 protein, which may include anablation or elimination of the protein. In some embodiments, themutation may be an amino substitution such as K51R in the NS2 protein.

Various features can be introduced into RSV strains of the presentinvention that change the characteristics of the virus in ways otherthan attenuation. For instance, codon optimization of the ORFs encodingthe proteins may be performed. Major protective antigens F and G canresult in increased antigen synthesis. The F and/or G protein gene maybe shifted upstream (closer to the promoter) to increase expression. TheF and/or G protein amino acid sequences can be modified to representcurrently-circulating strains, which can be particularly important inthe case of the divergent G protein, or to represent early-passageclinical isolates. Deletions or substitutions may be introduced into theG protein to obtain improved immunogenicity or other desired properties.For example, the CX3C fractalkine motif in the G protein might beablated to improve immunogenicity (Chirkova et al. J Virol 201387:13466-13479).

For example, in some embodiments, the nucleotide sequence encoding the Gprotein of the RSV may be replaced with a nucleotide sequence from theclinical isolate A/Maryland/001/11. In some embodiments, the nucleotidesequence encoding the F protein of the RSV may be replaced with anucleotide sequence from the clinical isolate A/Maryland/001/11, e.g.F001.

In some embodiments, a native or naturally occurring nucleotide sequenceencoding a protein of the RSV may be replaced with a codon optimizedsequence designed for increased expression in a selected host, inparticular the human. For example, in some embodiments, the nucleotidesequence encoding the F protein of the RSV may be replaced with a codonoptimized sequence. In some embodiments, the nucleotide sequenceencoding the F protein of the RSV may be replaced with the codonoptimized sequence from the clinical isolate A/Maryland/001/11. In someembodiments, the nucleotide sequence encoding the G protein of the RSVmay be replaced with the codon optimized nucleotide sequence from theclinical isolate A/Maryland/001/11.

Yet additional aspects of the invention involve changing the position ofa gene or altering gene order. For example, the NS1, NS2, SH and G genesmay be deleted individually, or the NS1 and NS2 gene may be deletedtogether, thereby shifting the position of each downstream gene relativeto the viral promoter. For example, when NS1 and NS2 are deletedtogether, N is moved from gene position 3 to gene position 1, P fromgene position 4 to gene position 2, and so on. Alternatively, deletionof any other gene within the gene order will affect the position(relative to the promoter) only of those genes which are located furtherdownstream. For example, SH occupies position 6 in Wild type virus, andits deletion does not affect M at position 5 (or any other upstreamgene) but moves G from position 7 to 6 relative to the promoter. Itshould be noted that gene deletion also can occur (rarely) in abiologically-derived mutant virus. For example, a subgroup B RSV thathad been passaged extensively in cell culture spontaneously deleted theSH and G genes (Karron et al. Proc. Natl. Acad. Sci. USA 94:13961 13966,1997; incorporated herein by reference).

Gene order shifting modifications (i.e., positional modifications movingone or more genes to a more promoter-proximal or promoter-distallocation in the recombinant viral genome) result in viruses with alteredbiological properties. For example, RSV lacking NS1, NS2, SH,G, NS1 andNS2 together, or SH and G together, have been shown to be attenuated invitro, in vivo, or both. In particular, the G and F genes may beshifted, singly and in tandem, to a more promoter-proximal positionrelative to their wild-type gene order. These two proteins normallyoccupy positions 7 (G) and 8 (F) in the RSV gene order(NS1-NS2-N-P-M-SH-G-FM2-L). In some embodiments, the order of thenucleotide sequences encoding the G and the F proteins may be reversedrelative to the naturally occurring order.

In addition to the above described mutations, the attenuated virusesaccording to the invention can incorporate heterologous, coding ornon-coding nucleotide sequences from any RSV or RSV-like virus, e.g.,human, bovine, ovine, murine (pneumonia virus of mice), or avian (turkeyrhinotracheitis virus) pneumovirus, or from another enveloped virus, e.g., parainfluenza virus (PIV). Exemplary heterologous sequences includeRSV sequences from one human RSV strain combined with sequences from adifferent human RSV strain. Alternatively, the RSV may incorporatesequences from two or more, wild-type or mutant human RSV subgroups, forexample a combination of human RSV subgroup A and subgroup B sequences.In yet additional aspects, one or more human RSV coding or non-codingpolynucleotides are substituted with a counterpart sequence from aheterologous RSV or non-RSV virus to yield novel attenuated vaccinestrains.

In addition to the recombinant RSVs having the particular mutations, andthe combinations of those mutations, described herein, the disclosedviruses may be modified further as would be appreciated by those skilledin the art. For example, the recombinant RSVs may have one or more ofits proteins deleted or otherwise mutated or a heterologous gene from adifferent organism may be added to the genome or antigenome so that therecombinant RSV expresses or incorporates that protein upon infecting acell and replicating. Furthermore, those skilled in the art willappreciate that other previously defined mutations known to have aneffect on RSV may be combined with one or more of any of the mutationsdescribed herein to produce a recombinant RSV with desirable attenuationor stability characteristics.

In some embodiments, the mutations described herein, when used eitheralone or in combination with another mutation, may provide for differentlevels of virus attenuation, providing the ability to adjust the balancebetween attenuation and immunogenicity, and provide a more stablegenotype than that of the parental virus.

Additional representative viruses from those described in thisdisclosure may be evaluated in cell culture for infectivity, replicationkinetics, yield, efficiency of protein expression, and genetic stabilityusing the methods described herein and illustrated in examples usingexemplary recombinant strains. Additional representative strains may beevaluated in rodents and non-human primates for infectivity, replicationkinetics, yield, immunogenicity, and genetic stability. While thesesemi-permissive systems may not reliably detect every difference inreplication, substantial differences in particular may be detected. Alsorecombinant strains may be evaluated directly in seronegative childrenwithout the prior steps of evaluation in adults and seropositivechildren. This may be done, for example, in groups of 10 vaccinerecipients and 5 placebo recipients, which is a small number that allowssimultaneous evaluation of multiple candidates. Candidates may beevaluated in the period immediately post-immunization for vaccine virusinfectivity, replication kinetics, shedding, tolerability,immunogenicity, and genetic stability, and the vaccines may be subjectedto surveillance during the following RSV season for safety, RSV disease,and changes in RSV-specific serum antibodies, as described in Karron, etal. 2015, Science Transl Med 2015 7(312):312ra175, which is incorporatedherein in its entirety. Thus, analysis of selected representativeviruses may provide for relatively rapid triage to narrow downcandidates to identify the most optimal.

Reference to a protein or a peptide includes its naturally occurringform, as well as any fragment, domain, or homolog of such protein. Asused herein, the term “homolog” is used to refer to a protein or peptidewhich differs from a naturally occurring protein or peptide (i.e., the“prototype” or “wild-type” protein) by minor modifications to thenaturally occurring protein or peptide, but which maintains the basicprotein and side chain structure of the naturally occurring form. Suchchanges include, but are not limited to: changes in one or a few aminoacid side chains; changes in one or a few amino acids, includingdeletions (e.g., a truncated version of the protein or peptide)insertions and/or substitutions; changes in stereochemistry of one or afew atoms; and/or minor derivatizations, including but not limited to:methylation, glycosylation, phosphorylation, acetylation,myristoylation, prenylation, palmitation, amidation. A homolog can haveeither enhanced, decreased, or substantially similar properties ascompared to the naturally occurring protein or peptide. A homolog of agiven protein may comprise, consist essentially of, or consist of, anamino acid sequence that is at least about 50%, or at least about 55%,or at least about 60%, or at least about 65%, or at least about 70%, orat least about 75%, or at least about 80%, or at least about 85%, or atleast about 90%, or at least about 95%, or at least about 96%, or atleast about 97%, or at least about 98%, or at least about 99% identical(or any percent identity between 45% and 99%, in whole integerincrements), to the amino acid sequence of the reference protein.

In one aspect of the invention, a selected gene segment, such as oneencoding a selected protein or protein region (e.g., a cytoplasmic tail,transmembrane domain or ectodomain, an epitopic site or region, abinding site or region, an active site or region containing an activesite, etc.) from one RSV, can be substituted for a counterpart genesegment from the same or different RSV or other source, to yield novelrecombinants having desired phenotypic changes compared to wild-type orparent RSV strains. For example, recombinants of this type may express achimeric protein having a cytoplasmic tail and/or transmembrane domainof one RSV fused to an ectodomain of another RSV. Other exemplaryrecombinants of this type express duplicate protein regions, such asduplicate immunogenic regions. As used herein, “counterpart” genes, genesegments, proteins or protein regions, are typically from heterologoussources (e.g., from different RSV genes, or representing the same (i.e.,homologous or allelic) gene or gene segment in different RSV strains).Typical counterparts selected in this context share gross structuralfeatures, e.g., each counterpart may encode a comparable structural“domain,” such as a cytoplasmic domain, transmembrane domain,ectodomain, binding site or region, epitopic site or region, etc.Counterpart domains and their encoding gene segments embrace anassemblage of species having a range of size and amino acid (ornucleotide) sequence variations, which range is defined by a commonbiological activity among the domain or gene segment variants. Forexample, two selected protein domains encoded by counterpart genesegments within the invention may share substantially the samequalitative activity, such as providing a membrane spanning function, aspecific binding activity, an immunological recognition site, etc. Moretypically, a specific biological activity shared between counterparts,e.g., between selected protein segments or proteins, will besubstantially similar in quantitative terms, i.e., they will not vary inrespective quantitative activity profiles by more than 30%, preferablyby no more than 20%, more preferably by no more than 5-10%.

In alternative aspects of the invention, the infectious RSV producedfrom a cDNA-expressed genome or antigenome can be any of the RSV orRSV-like strains, e.g., human, bovine, murine, etc., or of anypneumovirus or metapneumovirus, e.g., pneumonia virus of mice or avianmetapneumovirus. To engender a protective immune response, the RSVstrain may be one which is endogenous to the subject being immunized,such as human RSV being used to immunize humans. The genome orantigenome of endogenous RSV can be modified, however, to express RSVgenes or gene segments from a combination of different sources, e.g., acombination of genes or gene segments from different RSV species,subgroups, or strains, or from an RSV and another respiratory pathogensuch as human parainfluenza virus (PIV) (see, e.g., Hoffman et al. J.Virol. 71:4272-4277 (1997); Durbin et al. Virology 235(2):323-32 (1997);Murphy et al. U.S. Patent Application Ser. No. 60/047,575, filed May 23,1997, and the following plasmids for producing infectious PIV clones:p3/7(131) (ATCC 97990); p3/7(131)2G(ATCC 97889); and p218(131) (ATCC97991); each deposited Apr. 18, 1997 under the terms of the BudapestTreaty with the American Type Culture Collection (ATCC) of 10801University Blvd., Manassas, Va. 20110-2209, USA., and granted the aboveidentified accession numbers.

In certain embodiments of the invention, recombinant RSV are providedwherein individual internal genes of a human RSV are replaced with,e.g., a bovine or other RSV counterpart, or with a counterpart orforeign gene from another respiratory pathogen such as PIV.Substitutions, deletions, etc. of RSV genes or gene segments in thiscontext can include part or all of one or more of the NS1, NS2, N, P, M,SH, and L genes, or the M2-1 open reading frames, or non-immunogenicparts of the G and F genes. Also, human RSV cis-acting sequences, suchas promoter or transcription signals, can be replaced with, e.g., theirbovine RSV counterpart. Reciprocally, means are provided to generatelive attenuated bovine RSV by inserting human attenuating genes orcis-acting sequences into a bovine RSV genome or antigenome background.

Thus, infectious recombinant RSV intended for administration to humanscan be a human RSV that has been modified to contain genes from, e.g., abovine RSV or a PIV, such as for the purpose of attenuation. Forexample, by inserting a gene or gene segment from PIV, a bivalentvaccine to both PIV and RSV is provided. Alternatively, a heterologousRSV species, subgroup or strain, or a distinct respiratory pathogen suchas PIV, may be modified, e.g., to contain genes that encode epitopes orproteins which elicit protection against human RSV infection. Forexample, the human RSV glycoprotein genes can be substituted for thebovine glycoprotein genes such that the resulting bovine RSV, which nowbears the human RSV surface glycoproteins and would retain a restrictedability to replicate in a human host due to the remaining bovine geneticbackground, elicits a protective immune response in humans against humanRSV strains.

The ability to analyze and incorporate other types of attenuatingmutations into infectious RSV for vaccine development extends to a broadassemblage of targeted changes in RSV clones. For example, any RSV genewhich is not essential for growth may be ablated or otherwise modifiedto yield desired effects on virulence, pathogenesis, immunogenicity andother phenotypic characters. In addition, a variety of other geneticalterations can be produced in a recombinant RSV genome or antigenomefor incorporation into infectious recombinant RSV, alone or togetherwith one or more attenuating point mutations adopted from a biologicallyderived mutant RSV.

As used herein, “heterologous genes” refers to genes taken fromdifferent RSV strains or types or non-RSV sources. These heterologousgenes can be inserted in whole or in part, the order of genes changed,gene overlap removed, the RSV genome promoter replaced with itsantigenome counterpart, portions of genes removed or substituted, andeven entire genes deleted. Different or additional modifications in thesequence can be made to facilitate manipulations, such as the insertionof unique restriction sites in various intergenic regions (e.g., aunique Stul site between the G and F genes) or elsewhere. Nontranslatedgene sequences can be removed to increase capacity for inserting foreignsequences.

Deletions, insertions, substitutions and other mutations involvingchanges of whole viral genes or gene segments in recombinant RSV of theinvention yield highly stable vaccine candidates, which are particularlyimportant in the case of immunosuppressed individuals. Many of thesemutations will result in attenuation of resultant vaccine strains,whereas others will specify different types of desired phenotypicchanges. For example, certain viral genes are known which encodeproteins that specifically interfere with host immunity (see, e.g., Katoet al., EMBO. J. 16:578-87 (1997). Ablation of such genes in vaccineviruses is expected to reduce virulence and pathogenesis and/or improveimmunogenicity.

Other mutations within RSV of the present invention involve replacementof the 3′ end of genome with its counterpart from antigenome, which isassociated with changes in RNA replication and transcription. Inaddition, the intergenic regions (Collins et al., Proc. Natl. Acad. Sci.USA 83:4594-4598 (1986)) can be shortened or lengthened or changed insequence content, and the naturally-occurring gene overlap (Collins etal., Proc. Natl. Acad. Sci. USA 84:5134-5138 (1987)) can be removed orchanged to a different intergenic region by the methods describedherein.

In another embodiment, a sequence surrounding a translational start site(preferably including a nucleotide in the −3 position) of a selected RSVgene is modified, alone or in combination with introduction of anupstream start codon, to modulate RSV gene expression by specifying up-or down-regulation of translation.

Alternatively, or in combination with other RSV modifications disclosedherein, RSV gene expression can be modulated by altering atranscriptional GS signal of a selected gene(s) of the virus. In oneexemplary embodiment, the GS signal of NS2 is modified to include adefined mutation to superimpose a is restriction on viral replication.

Yet additional RSV clones within the invention incorporate modificationsto a transcriptional GE signal. For example, RSV clones are providedwhich substitute or mutate the GE signal of the NS1 and NS2 genes forthat of the N gene, resulting in decreased levels of readthrough mRNAsand increased expression of proteins from downstream genes. Theresulting recombinant virus exhibits increased growth kinetics andincreased plaque size, providing but one example of alteration of RSVgrowth properties by modification of a cis-acting regulatory element inthe RSV genome.

In another aspect, expression of the G protein may be increased bymodification of the G mRNA. The G protein is expressed as both amembrane bound and a secreted form, the latter form being expressed bytranslational initiation at a start site within the G gene translationalopen reading frame. The secreted form may account for as much asone-half of the expressed G protein. Ablation of the internal start site(e.g., by sequence alteration, deletion, etc.), alone or together withaltering the sequence context of the upstream start site yields desiredchanges in G protein expression. Ablation of the secreted form of the Gprotein also will improve the quality of the host immune response toexemplary, recombinant RSV, because the soluble form of the G protein isthought to act as a “decoy” to trap neutralizing antibodies. Also,soluble G protein has been implicated in enhanced immunopathology due toits preferential stimulation of a Th2-biased response.

In related aspects, levels of RSV gene expression may be modified at thelevel of transcription. In one aspect, the position of a selected genein the RSV gene map may be changed to a more promoter-proximal orpromoter-distal position, whereby the gene will be expressed more orless efficiently, respectively. According to this aspect, modulation ofexpression for specific genes can be achieved yielding reductions orincreases of gene expression from two-fold, more typically four-fold, upto ten-fold or more compared to wild-type levels. In one example, theNS2 gene (second in order in the RSV gene map) is substituted inposition for the SH gene (sixth in order), yielding a predicted decreasein expression of NS2. Increased expression of selected RSV genes due topositional changes can be achieved up to 10-fold, 30-fold, 50-fold,100-fold or more, often attended by a commensurate decrease inexpression levels for reciprocally, positionally substituted genes.

In some exemplary embodiments, the F and G genes may be transpositionedsingly or together to a more promoter-proximal or promoter-distal sitewithin the (recombinant) RSV gene map to achieve higher or lower levelsof gene expression, respectively. These and other transpositioningchanges yield novel RSV clones having attenuated phenotypes, for exampledue to decreased expression of selected viral proteins involved in RNAreplication. In yet other embodiments, RSV useful in a vaccineformulation may be conveniently modified to accommodate antigenic driftin circulating virus. Typically the modification will be in the G and/orF proteins. The entire G or F gene, or the segments encoding particularimmunogenic regions thereof, is incorporated into the RSV genome orantigenome cDNA by replacement of the corresponding region in theinfectious clone or by adding one or more copies of the gene such thatseveral antigenic forms are represented.

Progeny virus produced from the modified RSV cDNA are then used invaccination protocols against the emerging strains. Further, inclusionof the G protein gene of RSV subgroup B as a gene addition will broadenthe response to cover a wider spectrum of the relatively diversesubgroup A and B strains present in the human population.

An infectious RSV clone of the invention may also be engineeredaccording to the methods and compositions disclosed herein to enhanceits immunogenicity and induce a level of protection greater than thatprovided by infection with a wild-type RSV or an incompletely attenuatedparental virus or clone. For example, an immunogenic epitope from aheterologous RSV strain or type, or from a non-RSV source such as PIV,can be added by appropriate nucleotide changes in the polynucleotidesequence encoding the RSV genome or antigenome. Recombinant RSV can alsobe engineered to identify and ablate (e.g., by amino acid insertion,substitution or deletion) epitopes associated with undesirableimmunopathologic reactions. In other embodiments, an additional gene maybe inserted into or proximate to the RSV genome or antigenome which isunder the control of an independent set of transcription signals. Genesof interest may include, but are not limited to, those encodingcytokines (e.g., IL-2 through IL-15, especially IL-2, IL-6 and IL-12,etc.), gamma-interferon, and include those encoding cytokines (e.g.,IL-2 through IL-15, especially IL-2, IL-6 and IL-12, etc.),gamma-interferon, and proteins rich in T helper cell epitopes. Theadditional protein can be expressed either as a separate protein or as achimera engineered from a second copy of one of the RSV proteins, suchas SH. This provides the ability to modify and improve the immuneresponse against RSV both quantitatively and qualitatively.

In addition to the above described modifications to recombinant RSV,different or additional modifications in RSV clones can be made tofacilitate manipulations, such as the insertion of unique restrictionsites in various intergenic regions (e.g., a unique Stul site betweenthe G and F genes) or elsewhere. Nontranslated gene sequences can beremoved to increase capacity for inserting foreign sequences.

Introduction of the foregoing, defined mutations into an infectious RSVclone can be achieved by a variety of well-known methods. By “infectiousclone” is meant cDNA or its product, synthetic or otherwise, which canbe transcribed into genomic or antigenomic RNA capable of producing aninfectious virus. The term “infectious” refers to a virus or viralstructure that is capable of replicating in a cultured cell or animal orhuman host to produce progeny virus or viral structures capable of thesame activity. Thus, defined mutations can be introduced by conventionaltechniques (e.g., site-directed mutagenesis) into a cDNA copy of thegenome or antigenome. The use of antigenome or genome cDNA subfragmentsto assemble a complete antigenome or genome cDNA is well-known by thoseof ordinary skill in the art and has the advantage that each region canbe manipulated separately (smaller cDNAs are easier to manipulate thanlarge ones) and then readily assembled into a complete cDNA. Thus, thecomplete antigenome or genome cDNA, or any subfragment thereof, can beused as template for oligonucleotide-directed mutagenesis. A mutatedsubfragment can then be assembled into the complete antigenome or genomecDNA. Mutations can vary from single nucleotide changes to replacementof large cDNA pieces containing one or more genes or genome regions.

Recombinant RSV may be produced by the intracellular coexpression of acDNA that encodes the RSV genomic RNA, together with those viralproteins necessary to generate a transcribing, replicating nucleocapsid.Plasmids encoding other RSV proteins may also be included with theseessential proteins. Alternatively, RNA may be synthesized in in vitrotranscription reactions and transfected into cultured cells.

Accordingly, also described herein are isolated polynucleotides thatencode the described mutated viruses, make up the described genomes orantigenomes, express the described genomes or antigenomes, or encodevarious proteins useful for making recombinant RSV in vitro.Polynucleotides comprising the sequences of any of the SEQ ID NOsdescribed herein are included in the present invention. Further includedare polynucleotides comprising sequences that consist or consistessentially of any of the aforementioned sequences, sequences thatpossess at least about 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81,82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99or 100 percent identity (or any percent identity in between) to any ofthe aforementioned SEQ ID NOs, as well as polynucleotides that hybridizeto, or are the complements of the aforementioned molecules.

These polynucleotides can be included within or expressed by vectors inorder to produce a recombinant RSV. Accordingly, cells transfected withthe isolated polynucleotides or vectors are also within the scope of theinvention and are exemplified herein. Thus, in some embodiments, thepresent invention includes a vector comprising the isolatedpolynucleotide molecules described above. In some embodiments, thepresent invention includes a cell comprising the isolated polynucleotidemolecules described above.

In related aspects of the invention, compositions (e.g., isolatedpolynucleotides and vectors incorporating an RSV-encoding cDNA) andmethods are provided for producing an isolated infectious recombinantRSV bearing an attenuating mutation. Included within these aspects ofthe invention are novel, isolated polynucleotide molecules and vectorsincorporating such molecules that comprise a RSV genome or antigenomewhich is modified as described herein. Also provided is the same ordifferent expression vector comprising one or more isolatedpolynucleotide molecules encoding the RSV proteins. These proteins alsocan be expressed directly from the genome or antigenome cDNA. Thevector(s) is/are preferably expressed or coexpressed in a cell orcell-free lysate, thereby producing a mutant RSV particle or subviralparticle.

In one aspect, the invention includes a method for producing one or morepurified RSV protein(s) is provided which involves infecting a host cellpermissive of RSV infection with a recombinant RSV strain underconditions that allow for RSV propagation in the infected cell. After aperiod of replication in culture, the cells are lysed and recombinantRSV is isolated therefrom. One or more desired RSV protein(s) ispurified after isolation of the virus, yielding one or more RSVprotein(s) for vaccine, diagnostic and other uses.

The above methods and compositions for producing attenuated recombinantRSV mutants yield infectious viral or subviral particles, or derivativesthereof An infectious virus is comparable to the authentic RSV virusparticle and is infectious as is. It can directly infect fresh cells. Aninfectious subviral particle typically is a subcomponent of the virusparticle which can initiate an infection under appropriate conditions.For example, a nucleocapsid containing the genomic or antigenomic RNAand the N, P, L and M2-1 proteins is an example of a subviral particlewhich can initiate an infection if introduced into the cytoplasm ofcells. Subviral particles provided within the invention include viralparticles which lack one or more protein(s), protein segment(s), orother viral component(s) not essential for infectivity.

In other embodiments the invention provides a cell or cell free lysatecontaining an expression vector which comprises an isolatedpolynucleotide molecule encoding attenuated recombinant RSV genome orantigenome as described above, and an expression vector (the same ordifferent vector) which comprises one or more isolated polynucleotidemolecules encoding the N, P, L and RNA polymerase elongation factorproteins of RSV. One or more of these proteins also can be expressedfrom the genome or antigenome cDNA. Upon expression the genome orantigenome and N, P, L, and RNA polymerase elongation factor proteinscombine to produce an infectious RSV viral or sub-viral particle.

The recombinant RSV of the invention are useful in various compositionsto generate a desired immune response against RSV in a host susceptibleto RSV infection. Attenuated rRSV strains of the invention are capableof eliciting a protective immune response in an infected human host, yetare sufficiently attenuated so as to not cause unacceptable symptoms ofsevere respiratory disease in the immunized host. The attenuated virusor subviral particle may be present in a cell culture supernatant,isolated from the culture, or partially or completely purified. Thevirus may also be lyophilized, and can be combined with a variety ofother components for storage or delivery to a host, as desired.

In another aspect of the invention, the recombinant RSV strains may beemployed as “vectors” for protective antigens of other pathogens,particularly respiratory tract pathogens such as parainfluenza virus(PIV). For example, recombinant RSV having a T11661 mutation may beengineered which incorporate, sequences that encode protective antigensfrom PIV to produce infectious, attenuated vaccine virus.

In some embodiments, the invention includes a pharmaceutical compositioncomprising an immunologically effective amount of the recombinant RSVvariant encoded by the isolated polynucleotide molecules describedabove. In some embodiments, the invention includes a method ofvaccinating a subject or a method of inducing an immune responsecomprising administering the pharmaceutical composition. The compositionmay be administered by any suitable method, including but not limitedto, via injection, aerosol delivery, nasal spray, nasal droplets, oralinoculation, or topical application. In some embodiments, it may beadministered by, via injection, aerosol delivery, nasal spray, nasaldroplets. The composition may be administered intranasally orsubcutaneously or intramuscularly. In some embodiments, it may beadministered intranasally. The methods and routes of administration arefurther described in detail below.

In related aspects, the invention provides a method for stimulating theimmune system of an individual to elicit an immune response against RSVin a mammalian subject. The method comprises administering animmunogenic formulation of an immunologically sufficient or effectiveamount of an attenuated RSV in a physiologically acceptable carrierand/or adjuvant.

In some embodiments, the invention includes a live attenuated RSVvaccine comprising the recombinant RSV variant encoded by the isolatedpolynucleotide molecules described above. In some embodiments, theinvention includes a pharmaceutical composition comprising the RSVvaccine. In a related aspect, the invention includes a method of makinga vaccine comprising expressing the isolated polynucleotide moleculesdescribed above.

The vaccines may comprise a physiologically acceptable carrier and/oradjuvant and an isolated attenuated recombinant RSV particle or subviralparticle. In some embodiments, the vaccine is comprised of an attenuatedrecombinant RSV having at least one and preferably two or more mutationsdescribed herein or other nucleotide modifications to achieve a suitablebalance of attenuation and immunogenicity.

To select candidate vaccine viruses from the host of recombinant RSVstrains provided herein, the criteria of viability, efficientreplication in vitro, attenuation in vivo, immunogenicity, andphenotypic stability are determined according to well-known methods.Viruses which will be most desired in vaccines of the invention mustmaintain viability, must replicate sufficiently in vitro well underpermissive conditions to make vaccine manufacture possible, must have astable attenuation phenotype, must be well-tolerated, must exhibitreplication in an immunized host (albeit at lower levels), and musteffectively elicit production of an immune response in a vaccinesufficient to confer protection against serious disease caused bysubsequent infection from wild-type virus. Clearly, the heretofore knownand reported RSV mutants do not meet all of these criteria. Indeed,contrary to expectations based on the results reported for knownattenuated RSV, viruses of the invention are not only viable and moreattenuated then previous mutants, but are more stable genetically invivo than those previously studied mutants.

To propagate a RSV virus for vaccine use and other purposes, a number ofcell lines which allow for RSV growth may be used. RSV grows in avariety of human and animal cells. Preferred cell lines for propagatingattenuated RS virus for vaccine use include DBSFRhL-2, MRC-5, and Verocells. Highest virus yields are usually achieved with epithelial celllines such as Vero cells. Cells are typically inoculated with virus at amultiplicity of infection ranging from about 0.001 to 1.0, or more, andare cultivated under conditions permissive for replication of the virus,e.g., at about 30-37° C. and for about 3-10 days, or as long asnecessary for virus to reach an adequate titer. Temperature-sensitiveviruses often are grown using 32° C. as the “permissive temperature.”Virus is removed from cell culture and separated from cellularcomponents, typically by well-known clarification procedures, e.g.,centrifugation, and may be further purified as desired using procedureswell known to those skilled in the art.

RSV which has been attenuated as described herein can be tested invarious well known and generally accepted in vitro and in vivo models toconfirm adequate attenuation, resistance to phenotypic reversion, andimmunogenicity for vaccine use. In in vitro assays, the modified virus,which can be a multiply attenuated, biologically derived or recombinantRSV, is tested for temperature sensitivity of virus replication or “tsphenotype,” and for the small plaque phenotype. Modified viruses arefurther tested in animal models of RSV infection. A variety of animalmodels (e.g., murine, cotton rat, and primate) have been described andare known to those skilled in the art.

In accordance with the foregoing description and based on the Examplesbelow, the invention also provides isolated, infectious RSV compositionsfor vaccine use. The attenuated virus which is a component of a vaccineis in an isolated and typically purified form. By isolated is meant torefer to RSV which is in other than a native environment of a wild-typevirus, such as the nasopharynx of an infected individual. Moregenerally, isolated is meant to include the attenuated virus as acomponent of a cell culture or other artificial medium. For example,attenuated RSV of the invention may be produced by an infected cellculture, separated from the cell culture and added to a stabilizer.

RSV vaccines of the invention contain as an active ingredient animmunogenically effective amount of RSV produced as described herein.Biologically derived or recombinant RSV can be used directly in vaccineformulations. The biologically derived or recombinantly modified virusmay be introduced into a host with a physiologically acceptable carrierand/or adjuvant. Useful carriers are well known in the art, and include,e.g., water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acidand the like. The resulting aqueous solutions may be packaged for use asis, or in frozen form that is thawed prior to use, or lyophilized, thelyophilized preparation being combined with a sterile solution prior toadministration, as mentioned above. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, which include, but are not limitedto, pH adjusting and buffering agents, tonicity adjusting agents,wetting agents and the like, for example, sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, sucrose,magnesium sulfate, phosphate buffers, HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, sorbitanmonolaurate, and triethanolamine oleate. Acceptable adjuvants includeincomplete Freund's adjuvant, aluminum phosphate, aluminum hydroxide, oralum, which are materials well known in the art. Preferred adjuvantsalso include Stimulon™ QS-21 (Aquila Biopharmaceuticals, Inc.,Worchester, Mass.), MPL™ (3-0-deacylated monophosphoryl lipid A; RIBIImmunoChem Research, Inc., Hamilton, Mont.), and interleukin-12(Genetics Institute, Cambridge, Mass.).

Upon immunization with a RSV vaccine composition, the host responds tothe vaccine by producing antibodies specific for RSV virus proteins,e.g., F and G glycoproteins. In addition, innate and cell-mediatedimmune responses are induced, which can provide antiviral effectors aswell as regulating the immune response. As a result of the vaccinationthe host becomes at least partially or completely immune to RSVinfection, or resistant to developing moderate or severe RSV disease,particularly of the lower respiratory tract.

The host to which the vaccine is administered can be any mammalsusceptible to infection by RSV or a closely related virus and capableof generating a protective immune response to antigens of thevaccinizing strain. Thus, suitable hosts include humans, non-humanprimates, bovine, equine, swine, ovine, caprine, lagamorph, rodents,such as mice or cotton rats, etc. Accordingly, the invention providesmethods for creating vaccines for a variety of human and veterinaryuses.

The vaccine compositions containing the attenuated RSV of the inventionare administered to a subject susceptible to or otherwise at risk of RSVinfection in an “immunogenically effective dose” which is sufficient toinduce or enhance the individual's immune response capabilities againstRSV. An RSV vaccine composition may be administered by any suitablemethod, including but not limited to, via injection, aerosol delivery,nasal spray, nasal droplets, oral inoculation, or topical application.In the case of human subjects, the attenuated virus of the invention isadministered according to well established human RSV vaccine protocols(Karron et al. HD 191:1093-104, 2005). Briefly, adults or children areinoculated intranasally via droplet with an immunogenically effectivedose of RSV vaccine, typically in a volume of 0.5 ml of aphysiologically acceptable diluent or carrier. This has the advantage ofsimplicity and safety compared to parenteral immunization with anon-replicating vaccine. It also provides direct stimulation of localrespiratory tract immunity, which plays a major role in resistance toRSV. Further, this mode of vaccination effectively bypasses theimmunosuppressive effects of RSV specific maternally-derived serumantibodies, which typically are found in the very young. Also, while theparenteral administration of RSV antigens can sometimes be associatedwith immunopathologic complications, this has never been observed with alive virus.

In some embodiments, the vaccine may be administered intranasally orsubcutaneously or intramuscularly. In some embodiments, it may beadministered to the upper respiratory tract. This may be performed byany suitable method, including but not limited to, by spray, droplet oraerosol delivery. Often, the composition will be administered to anindividual seronegative for antibodies to RSV or possessingtransplacentally acquired maternal antibodies to RSV.

In all subjects, the precise amount of RSV vaccine administered and thetiming and repetition of administration will be determined by variousfactors, including the patient's state of health and weight, the mode ofadministration, the nature of the formulation, etc. Dosages willgenerally range from about 3.0 log₁₀ to about 6.0 log₁₀ plaque formingunits (“PFU”) or more of virus per patient, more commonly from about 4.0log₁₀ to 5.0 log₁₀ PFU virus per patient. In one embodiment, about 5.0log₁₀ to 6.0 log₁₀ PFU per patient may be administered during infancy,such as between 1 and 6 months of age, and one or more additionalbooster doses could be given 2-6 months or more later. In anotherembodiment, young infants could be given a dose of about 5.0 log₁₀ to6.0 log₁₀ PFU per patient at approximately 2, 4, and 6 months of age,which is the recommended time of administration of a number of otherchildhood vaccines. In yet another embodiment, an additional boosterdose could be administered at approximately 10-15 months of age. In anyevent, the vaccine formulations should provide a quantity of attenuatedRSV of the invention sufficient to effectively stimulate or induce ananti-RSV immune response (an “effective amount”).

In some embodiments, the vaccine may comprise attenuated recombinant RSVvirus that elicits an immune response against a single RSV strain orantigenic subgroup, e.g., A or B, or against multiple RSV strains orsubgroups. In this regard, rRSV can be combined in vaccine formulationswith other RSV vaccine strains or subgroups having different immunogeniccharacteristics for more effective protection against one or multipleRSV strains or subgroups. They may be administered in a vaccine mixture,or administered separately in a coordinated treatment protocol, toelicit more effective protection against one RSV strain, or againstmultiple RSV strains or subgroups.

The resulting immune response can be characterized by a variety ofmethods. These include taking samples of nasal washes or sera foranalysis of RSV-specific antibodies, which can be detected by testsincluding, but not limited to, complement fixation, plaqueneutralization, enzyme-linked immunosorbent assay,luciferase-immunoprecipitation assay, and flow cytometry. In addition,immune responses can be detected by assay of cytokines in nasal washesor sera, ELISPOT of immune cells from either source, quantitative RT-PCRor microarray analysis of nasal wash or serum samples, and restimulationof immune cells from nasal washes or serum by re-exposure to viralantigen in vitro and analysis for the production or display ofcytokines, surface markers, or other immune correlates measured by flowcytometry or for cytotoxic activity against indicator target cellsdisplaying RSV antigens. In this regard, individuals are also monitoredfor signs and symptoms of upper respiratory illness.

In some embodiments, neonates and infants are given multiple doses ofRSV vaccine to elicit sufficient levels of immunity. Administration maybegin within the first month of life, and at intervals throughoutchildhood, such as at two months, four months, six months, one year andtwo years, as necessary to maintain sufficient levels of protectionagainst natural RSV infection. In other embodiments, adults who areparticularly susceptible to repeated or serious RSV infection, such as,for example, health care workers, day care workers, family members ofyoung children, the elderly, individuals with compromisedcardiopulmonary function, are given multiple doses of RSV vaccine toestablish and/or maintain protective immune responses. Levels of inducedimmunity can be monitored by measuring amounts of neutralizing secretoryand serum antibodies, and dosages adjusted or vaccinations repeated asnecessary to maintain desired levels of protection. Further, differentvaccine viruses may be indicated for administration to differentrecipient groups. For example, an engineered RSV strain expressing acytokine or an additional protein rich in T cell epitopes may beparticularly advantageous for adults rather than for infants. Vaccinesproduced in accordance with the present invention can be combined withviruses of the other subgroup or strains of RSV to achieve protectionagainst multiple RSV subgroups or strains, or selected gene segmentsencoding, e.g., protective epitopes of these strains can be engineeredinto one RSV clone as described herein. In such embodiments, thedifferent viruses can be in admixture and administered simultaneously orpresent in separate preparations and administered separately. Forexample, as the F glycoproteins of the two RSV subgroups differ by onlyabout 11% in amino acid sequence, this similarity is the basis for across-protective immune response as observed in animals immunized withRSV or F antigen and challenged with a heterologous strain. Thus,immunization with one strain may protect against different strains ofthe same or different subgroup.

The level of attenuation of vaccine virus may be determined by, forexample, quantifying the amount of virus present in the respiratorytract of an immunized host and comparing the amount to that produced bywild-type RSV or other attenuated RS viruses which have been evaluatedas candidate vaccine strains. For example, the attenuated virus of theinvention will have a greater degree of restriction of replication inthe upper respiratory tract of a highly susceptible host, such as achimpanzee, compared to the levels of replication of wild-type virus,e.g., 10- to 1000-fold less. In order to further reduce the developmentof rhinorrhea, which is associated with the replication of virus in theupper respiratory tract, an ideal vaccine candidate virus should exhibita restricted level of replication in both the upper and lowerrespiratory tract. However, the attenuated viruses of the invention mustbe sufficiently infectious and immunogenic in humans to conferprotection in vaccinated individuals. Methods for determining levels ofRSV in the nasopharynx of an infected host are well known in theliterature. Specimens are obtained by aspiration or washing out ofnasopharyngeal secretions and virus quantified in tissue culture orother by laboratory procedure. See, for example, Belshe et al., J. Med.Virology 1:157-162 (1977), Friedewald et al., J. Amer. Med. Assoc.204:690-694 (1968); Gharpure et al., J. Virol. 3:414-421 (1969); andWright et al., Arch. Ges. Virusforsch. 41:238-247 (1973). The virus canconveniently be measured in the nasopharynx of host animals, such aschimpanzees.

The invention also provides methods for producing an infectious RSV fromone or more isolated polynucleotides, e.g., one or more cDNAs. Accordingto the present invention cDNA encoding a RSV genome or antigenome isconstructed for intracellular or in vitro coexpression with thenecessary viral proteins to form infectious RSV. By “RSV antigenome” ismeant an isolated positive-sense polynucleotide molecule which serves asthe template for the synthesis of progeny RSV genome. Preferably a cDNAis constructed which is a positive-sense version of the RSV genome,corresponding to the replicative intermediate RNA, or antigenome, so asto minimize the possibility of hybridizing with positive-sensetranscripts of the complementing sequences that encode proteinsnecessary to generate a transcribing, replicating nucleocapsid, i.e.,sequences that encode N, P, Land M2-1 protein.

A native RSV genome typically comprises a negative-sense polynucleotidemolecule which, through complementary viral mRNAs, encodes elevenspecies of viral proteins, i.e., the nonstructural proteins NS1 and NS2,N, P, matrix (M), small hydrophobic (SH), glycoprotein (G), fusion (F),M2-1, M2-2, and L, substantially as described in Mink et al., Virology185: 615-624 (1991), Stec et al., Virology 183: 273-287 (1991), andConnors et al., Virol. 208:478-484 (1995). For purposes of the presentinvention the genome or antigenome of the recombinant RSV of theinvention need only contain those genes or portions thereof necessary torender the viral or subviral particles encoded thereby infectious.Further, the genes or portions thereof may be provided by more than onepolynucleotide molecule, i.e., a gene may be provided by complementationor the like from a separate nucleotide molecule.

By recombinant RSV is meant a RSV or RSV-like viral or subviral particlederived directly or indirectly from a recombinant expression system orpropagated from virus or subviral particles produced therefrom. Therecombinant expression system will employ a recombinant expressionvector which comprises an operably linked transcriptional unitcomprising an assembly of at least a genetic element or elements havinga regulatory role in RSV gene expression, for example, a promoter, astructural or coding sequence which is transcribed into RSV RNA, andappropriate transcription initiation and termination sequences.

To produce infectious RSV from cDNA-expressed genome or antigenome, thegenome or antigenome is coexpressed with those RSV proteins necessary to(i) produce a nucleocapsid capable of RNA replication, and (ii) renderprogeny nucleocapsids competent for both RNA replication andtranscription. Transcription by the genome nucleocapsid provides theother RSV proteins and initiates a productive infection. Additional RSVproteins needed for a productive infection can also be supplied bycoexpression.

Alternative means to construct cDNA encoding the genome or antigenomeinclude by reverse transcription-PCR using improved PCR conditions(e.g., as described in Cheng et al., Proc. Natl. Acad. Sci. USA 91:5695-5699 (1994); Samal et al., J. Virol 70:5075-5082 (1996)) to reducethe number of subunit cDNA components to as few as one or two pieces. Inother embodiments, different promoters can be used (e.g., T3, SP6) ordifferent ribozymes (e.g., that of hepatitis delta virus). Different DNAvectors (e.g., cosmids) can be used for propagation to betteraccommodate the large size genome or antigenome.

The N, P, L and M2-1 proteins may be encoded by one or more expressionvectors which can be the same or separate from that which encodes thegenome or antigenome, and various combinations thereof. Additionalproteins may be included as desired, encoded by its own vector or by avector encoding a N, P, L, or M2-1 protein or the complete genome orantigenome. Expression of the genome or antigenome and proteins fromtransfected plasmids can be achieved, for example, by each cDNA beingunder the control of a promoter for T7 RNA polymerase, which in tum issupplied by infection, transfection or transduction with an expressionsystem for the T7 RNA polymerase, e.g., a vaccinia virus MVA strainrecombinant which expresses the T7 RNA polymerase (Wyatt et al.,Virology, 210:202-205 (1995)). The viral proteins, and/or T7 RNApolymerase, can also be provided from transformed mammalian cells, or bytransfection of preformed mRNA or protein.

In summary, the materials, information, and methods described in thisdisclosure provide an array of attenuated strains with gradedattenuation phenotypes, and provide guidance in selecting suitablevaccine candidate strains based on clinical benchmarks.

While various embodiments of the present invention have been describedin detail, it is apparent that modifications and adaptations of thoseembodiments will occur to those skilled in the art. It is to beexpressly understood, however, that such modifications and adaptationsare within the scope of the present invention, as set forth in thefollowing claims. The examples below are provided for the purpose ofillustration and are not intended to limit the scope of the presentinvention.

Each publication, sequence or other reference disclosed below andelsewhere herein is incorporated herein by reference in its entirety, tothe extent that there is no inconsistency with the present disclosure.The disclosures of U.S. Provisional Application 62/399,133 filed Sep.23, 2016 entitled Improved codon-pair-deoptimized vaccine candidates forhuman respiratory syncytial virus; U.S. Provisional Application62/400,476 filed Sep. 27, 2016 entitled Vaccine candidates forrespiratory syncytial virus (RSV) having attenuated phenotypes; andPublished U.S. Application US 2015-0368622 entitled Attenuation of humanrespiratory syncytial virus by genome scale codon-pair deoptimizationare incorporated in their entirety by reference.

EXAMPLES Materials and Methods

The following materials and methods were used in the examples below.

Virus harvest and titration. Vero cells were scraped into the tissueculture medium, vortexed for 30 sec, clarified by low speedcentrifugation, and snap-frozen. Virus titers in the clarified fluidswere determined by immunoplaque assay on Vero cells at 32° C.

Virus stocks were generated by scraping infected cells into media,followed by vortexing for 30 sec, clarification of the supernatant bycentrifugation. Virus aliquots were snap frozen and stored at −80° C.Virus titers were determined by plaque assay on Vero cells with an 0.8%methylcellulose overlay. After a 10 to 12-day incubation at 32° C.,plates were fixed with 80% cold methanol, and plaques were visualized byimmunostaining with a cocktail of three RSV-specific monoclonalantibodies. Titers were expressed as pfu per ml. Viral RNA was isolatedfrom all virus stocks, and sequence analysis of the viral genomes wasperformed from overlapping RT-PCR fragments by Sanger sequencing,confirming that the genomic sequences of the recombinant viruses werecorrect and free of adventitious mutations. The only sequences that werenot directly confirmed for each genome were the positions of theouter-most primers, namely nucleotides 1-23 and 15,174-15,222.

Ion torrent deep sequencing. Purified viral RNA from clarified culturefluids was copied into 8 overlapping fragments spanning the viralgenome. Libraries were prepared following the Ion torrent protocol,loaded into a semiconductor sequencing chip, and sequenced on a PersonalGenome Machine (Ion Torrent). A nucleotide variant was called if itoccurred >50 times with an average read depth of 1000× and aP-value<10-7 (Quality score >70).

Viral RNAs were extracted using the Qiagen Viral RNA extraction kit fromthe indicated aliquots of viruses that were passed during thetemperature stress test of Min_L or Min_FLC. Viral RNAs were reversetranscribed using the superscript II RT (Life Technologies) followingthe manufacturer recommendations. Then, the cDNAs were amplified by PCRusing RSV specific primers and the pfx DNA polymerase enzyme (LifeTechnologies) in eight overlapping fragments that cover the whole viralgenome. Each PCR product was purified using the QIAquick PCRpurification kit (Qiagen).

Equal amounts of DNA from each of the eight PCR reactions were pooledinto a 1.5 ml LoBind tube (Eppendorf). The DNA was subjected toenzymatic shearing using the ShearEnzyme (Ion Torrent) for 30 min at 37°C. in a heat block. The sheared DNA was then purified using 1.8 volumesof Agencourt magnetic beads (Beckman). The Agencourt beads were washedtwice with 0.2 ml of 70% ethanol, air dried for 5 min, and re-suspendedwith 20-30 μl of 10 mM Tris-HCl pH7.5 buffer followed by incubation atroom temperature for 5 min. DNA was recovered in the supernatant byplacing the 1.5 ml LoBind tube containing the Agencourt beads on amagnetic rack for 2 minutes (min). The DNA was treated withend-repairing enzyme (Ion Torrent) according to the manufacturer'sinstructions. The end-repaired DNA was purified with 1.8 volumes ofAgencourt beads and recovered in a magnetic rack as described above.

Approximately 100 ng of repaired DNA from each sample were used toligate with a specific barcode adapter and a sequencing adaptor in a 20μl reaction volume containing ligase and buffer (Ion Torrent) accordingto the manufacturer's instruction. The ligation reaction was carried outat room temperature for 30 min and terminated by adding 4 μl of 0.5MEDTA pH8.0. Equal volumes of different ligated DNA libraries were thencombined in a 1.5 ml LoBind tube and purified with 1.8 volumes ofAgencount and the DNA libraries were recovered as described above. TheDNA further underwent nick-translation using Bst 2.0 DNA polymerase andbuffer (NEB). Digested DNA was purified using a spin column MinElute kit(Qiagen).

Approximately 100 ng of the DNA libraries were then added into a PCR mixusing the Platinum High Fidelity DNA polymerase master mix (LifeTechnologies) followed by 2 cycles of PCR amplification at 95° C. for 10min followed by 2 cycles at 95° C. for 30 sec, 58° C. for 30 sec, and72° C. for 30 sec. The PCR products were further purified with 1.8volume of Agencourt and DNA was recovered using a magnetic rack asdescribed above. The DNA was quantified using the Qubit system(Invitrogen).

Approximately 70 million DNA molecules in 1 ml of PCR solution weremixed with a fixed ratio (0.5-1.0) of Ion sphere particles (ISP) (IonTorrent) in the presence of PCR reaction mix and oil (Ion Torrent) toform tens of millions of droplets of emulsion particles. These dropletswere passed through an enclosed capillary PCR plate in OneTouch (IonTorrent) which carried out the emulsion PCR amplification as the liquidand particles pass through the plate continuously. The ISPs wererecovered by centrifugation in OneTouch in a pair of collection tubes.At the end of the OneTouch emulsion PCR, the collection tubes werecentrifuged for 3 min at 15,000 g to remove most supernatant. The ISPswere washed once in 1 ml wash buffer (Ion Torrent) and centrifuged for 3min at 15,500 g to remove most supernatant. ISPs containing amplifiedDNA were further enriched from ISPs without DNA by incubating withDynabeads® MyOne™ Streptavidin Cl magnetic beads at room temperature for10 min in a rotating rack. The enriched IPSs were recovered by placingthe tube on a magnetic rack for 2 min, washed twice with 0.2 ml of washbuffer by pipetting and placing on a magnetic rack for 2 min and bydiscarding the supernatant. The ISPs were eluted from the Dynabeads®MyOne™ Streptavidin Cl magnetic beads by incubation with 0.4 ml 0.125NNaOH and 0.1% Tween 20 for 7 min at room temperature in a rotating rack.The eluted ISPs were washed twice with wash buffer and centrifuged for 4min at 15,500 g to remove most supernatant. The ISPs were resuspended bypipetting and placed on a magnetic rack for 2 min to remove last tracesof Dynabeads® MyOne™ beads.

100 μl of solution was transferred to a new tube as the final library ofISPs ready for QC testing and sequencing. For sequencing, the ISPs werecentrifuged for 3 min at 15,500 g to remove most of the supernatant. TheISPs were resuspended by pipetting and transferred into a 0.2 ml PCRtube containing 150 μl annealing buffer. Five microliter Control IonSpheres™ (Ion Torrent) was added to the ISPs mix and centrifuged for 3min at 15,500 g to remove most supernatant from the top to leave 15 μlat the bottom followed by adding 12 μl sequencing primer, denatured andannealed at 95° C. for 2 min, and 2 min at 37° C. 3 μl DNA polymerase(Ion Torrent) was added, and samples were loaded into a semiconductorsequencing chip 316 or 318 (Ion Torrent) to perform DNA sequencing on aPersonal Genome Machine (PGM) (Ion Torrent).

DNA sequences were analyzed against Min_FLC or Min_L reference sequencesusing VariantCaller 3.2 software from Ion Torrent on the Ion TorrentServer. The analysis pipeline was set at the default somatic variantconfiguration. A nucleotide variant was called if the variantoccurred >50 times with an average read depth of 1000× and aP-value<10⁻⁷ (Quality score>70) as previously described. The raw readdata were also manually verified using a genome browser IVG (The BroadInstitute).

Deep sequencing of long PCR fragments. Purified viral RNA from culturefluids was reverse transcribed using the Maxima H minus first strandcDNA synthesis kit (Thermo Scientific). Using RSV specific primers andthe SequalPrep long PCR kit (Life Technologies), the cDNAs were used togenerate a PCR product of 8.2 kb spanning the genome from the 3′end tothe middle of the M2-2 ORF. DNA template libraries were prepared,sequenced, and analyzed using PacBio kits and instrumentation and CluConsoftware.

The coexistence or not of mutations that arose in the M2-1 and P genesduring the first 4 passages of Min_L lineage #3 at 38 and 39° C. wasinvestigated by deep sequencing. To do so, viral RNAs from aliquots ofviruses derived from these passages were extracted using the Qiagenviral extraction kit as described above. Then, the viral RNAs werereverse transcribed using the Maxima H minus first strand cDNA synthesiskit (Thermo Scientific) following the manufacturer recommendations.Using RSV specific primers and the SequalPrep long PCR kit (lifetechnologies), cDNAs were then used to generate a PCR product of 8.2 kbthat covered a region from the 3′end of the genome to the M2-2 gene.

To prepare PacBio SMRTbell DNA template libraries, PCR products werepurified as described above and then concentrated using 0.45 volumes ofAMPure PB magnetic beads. To allow the DNA to bind to beads, the mixturewas mixed in a VWR vortex mixer at 2000 rpm for 10 min at roomtemperature. After a short spinning to pellet beads, each tube wasplaced in a magnetic bead rack and the supernatant was carefullydiscarded. Beads were then washed twice with 1.5 ml of freshly prepared70% ethanol. After removal of the ethanol, the bead pellet was allowedto dry for about 1 min. Then, the tube was removed from the magneticbead rack and centrifuged to pellet the beads. DNA was then eluted usingthe Pacific Biosciences Elution Buffer. To repair any DNA damage, theconcentrated DNA was incubated at 37° C. for 20 min in a LoBind tube inDNA damage repair buffer, NAD+, ATP high, dNTP and a DNA damage repairenzyme mix. Then, DNA was then incubated at 25° C. for 5 min with a DNAend repair mix. After the reaction, DNA was purified using AMPure PBbeads as described above, and eluted off the beads in 30 μl of elutionbuffer.

Then, end repaired DNA was annealed with a blunt end adapter in areaction containing the adapter, buffer, ATP and a ligase for 15 min at25° C. Ligase was inactivated at 65° C. for 10 min. Finally anexonuclease step for 1 h at 37° C. was included to remove any failedligation products. SMRTbell DNA template libraries were then purifiedthree times using AMPure PB beads as described above.

After purification, SMRTbell library templates were sequenced inSMRTcelis with the PacBio RSII instrument using the PacBio DNAPolymerase Binding Kit P6. Each sample library was sequenced on 2SMRTcells using MagBead loading and movie collection time of 240minutes.

Data were analyzed using CluCon software. All reads were aligned to thereference Min_L sequence. Only reads that span 99% of the full target(8161 bases or greater) were analyzed; yielding 32,738 near-full-lengthreads per time point on average (minimum 24,131 reads, maximum 41,118reads). The algorithm identifies variant positions by examining thealignments and finding positions where the minor frequencies observedcannot be explained statistically by chance noise. Fully-phasedhaplotypes are then estimated by tallying what was sequenced in each ofthe near-full length reads at the variant positions. A statistical testis used to discard “noisy” haplotypes or those that are simply explainedby other true observed haplotypes that have been corrupted by sequencingnoise.

Determination of the temperature shut-off of CPD rRSVs. The is phenotypeof each of the rRSV viruses was evaluated by efficiency of plaqueformation at 32, 35, 36, 37, 38, 39, and 40° C. Plaque assays wereperformed on Vero cells in duplicate, and incubated in sealed caskets atvarious temperatures in temperature controlled water-baths as previouslydescribed. The shut-off temperatures (TsH) is defined as the lowestrestrictive temperature at which there is a reduction in plaque numbercompared to 32° C. that is 100-fold or greater than that observed for wtRSV at the 2 temperatures.

Kinetics of virus replication in vitro. Multi-cycle and single cyclegrowth kinetics were performed on confluent monolayers of Vero cells atboth 32 and 37° C. in six-well plates.

In the multi-cycle growth kinetic experiments, Vero cells were infectedin duplicate at an MOI of 0.01 pfu/cell with the indicated viruses. Fromday 0 to 14, viruses were collected by scraping infected cells intomedia followed by vortexing for 30 sec, clarification of the supernatantby centrifugation. Virus inoculum and the daily aliquots were snapfrozen and stored at −80° C. Virus titers were determined by plaqueassay as described above.

In the single cycle growth kinetic experiments, three wells of Verocells in 6-well plates were infected at an MOI of 3 pfu/well at 32 or37° C. with the indicated viruses. Every four hours from four to 24 hpost-infection, cell-associated RNA was collected from one well usingthe RNeasy mini kit (Qiagen) following the manufacturer's instructions.For Western blot analysis, total cell lysates were collected in NuPageLDS sample buffer (Life Technologies) and then homogenized using aQIAshredder spin column (Qiagen). Finally, the last well was used tocollect virus and determined the virus titers, as described above.

Strand specific rRSV RNA quantification. Cell-associated RNA derivedfrom single cycle replication experiments was used to specificallyquantify viral negative sense (genomic) and positive sense (mRNA andantigenomic) RNA as described previously. qPCR results were analyzedusing the comparative threshold cycle (ACt) method, normalized to 18SrRNA, and then expressed as log2 fold increase over the indicatedreference sample.

Cell-associated RNA derived from single cycle replication experimentswas used to specifically quantify viral negative sense (genomic) andpositive sense (mRNA and antigenomic) RNA as described previously.Taqman assays for antigenomic/mRNA specific for each of the 11 wt RSVgenes were designed using Primer Express 3.0 software (LifeTechnologies). Specifically, for the L gene, three different taqmanassays were designed for the wt sequence and four for the CPD sequence.

One microgram of DNA-digest RNA was reverse transcribed usingSuperscript III (Life Technologies) in a 20 μl reaction using a taggedfirst strand primers, specific either to genome or to antigenomic/mRNA.After a five-fold dilution, each of the cDNAs was amplified intriplicate with a tag-specific primer, a second gene-specific primer,and a probe. Thus, only cDNAs containing the tagged RT primer sequencewere amplified. The probe sequence was RSV gene-specific. To normalizeresults, 18S rRNA was quantified in parallel using first strand cDNAgenerated with random primers, and a standard 18S rRNA taqman assay(Applied Biosystems). qPCR results were analyzed using the comparativethreshold cycle (ACt) method, normalized to 18S rRNA, and then expressedas loge fold increase over the Min_L 4 h time point, with the exceptionof wt L quantification, for which data was expressed as fold increaseover the wt 4 h time point. Negative controls without first strandprimer were included for each of the strand-specific qPCRs todemonstrate the absence of non-specific priming during first strand cDNAsynthesis.

Western blot analysis. Cell lysates prepared from single cycle infectionexperiments described above were separated on NuPAGE 4-12% Bis-TrisSDS-PAGE gels with MES electrophoresis buffer (Life Technologies) inparallel with Odyssey Two-Color Protein Molecular Weight Marker(Li-Cor). 30 μg of proteins were transferred to PVDF-F membranes(Millipore) in lx NuPAGE buffer. The membranes were blocked with Odysseyblocking buffer (LI-COR) and incubated with primary antibody in presenceof 0.1% Tween-20. The primary antibodies and the dilutions used were asfollows: mouse anti-RSV N, P, G, F and M2-1 monoclonal antibodies(1:1,000) were purchased from Abcam; rabbit polyclonal antiserum thatrecognized both NS1 and NS2 was generated by peptide immunization ofrabbits (Abgent) with a synthetic peptide representing the C-terminal 14amino acids of NS2 (the C-termini of NS2 and NS1 are identical for thelast 4 amino acids, which presumably accounts for the cross-reactivity);rabbit anti-GAPDH polyclonal antibody (1:200) used as loading control(Santa-Cruz Biotechnologies, Inc.). The secondary antibodies used at a1:15,000 dilution were goat anti-rabbit IgG IRDye 680 (Li-Cor) and goatanti-mouse IgG IRDye 800 (Li-Cor). Membranes were scanned on theOdyssey® Infrared Imaging System. Data was analyzed using Odysseysoftware, version 3.0 (Li-Cor). For quantification of identified RSVproteins of interest, fluorescence signals were background corrected.Values indicate the median fluorescence intensity of each protein band.

Determination of plaques sizes. Virus plaque sizes were determined byplaque assay on Vero cells using twenty-four well plates. Vero cellsmonolayers were inoculated with 30 pfu per well of previously titteredand sequenced virus stocks. After 2 h adsorption, a 0.8% methylcelluloseoverlay was added to each well. After a 12-day incubation at 32° C.,plates were fixed with 80% cold methanol. Then, wells were incubatedwith a cocktail of three RSV-specific monoclonal antibodies (Bukreyev etal. 2001) in blocking buffer (Odyssey buffer, Licor) for one hour. Afterwashing with blocking buffer, plaques were stained with goat anti-mouseIRdye 680LT (Licor) secondary antibody, and plaques were visualizedusing the Odyssey® Infrared Imaging System. Images were analyzed usingImage J and the area of more than 1000 plaques per virus was measuredand expressed in pixe12. Distribution of the virus plaque sizes wascompared for statistical significance using the Kolmogorov-Smirnov testfollowed by Bonferroni correction (Prism 6.0, GraphPad). Sets of datawere only considered statistically different at p<0.05.

Evaluation of the replication of CPD rRSVs in mice and hamsters. Animalstudies were approved by the NIAID Animal Care and Use Committee, andperformed using previously described methods.

All animal studies were approved by the National Institutes of Health(NIH) Institutional Animal Care and Use Committee (ACUC). Replication ofCPD viruses was evaluated in the upper and lower respiratory tract ofsix-week-old BALB/c mice as described previously. Group of 20 mice wereinoculated intranasally under isoflurane anesthesia with 10⁶ pfu of wtrRSV, Min_L, M2-1[A73S], M2-1[N88K] or NPM2-1[N88K]L. On days 4 and 5,eight mice from each group were sacrificed by carbon dioxide inhalation.The remaining four mice in each group were sacrificed on day 10. Nasalturbinates (NT) and lung tissues were harvested and homogenizedseparately in Leibovitz (L)-15 medium containing 1×SPG, 2% L-glutamine,0.06 mg/mL ciprofloxacin, 0.06 mg/mL clindamycin phosphate, 0.05 mg/mLgentamycin, and 0.0025 mg/mL amphotericin B. Virus titers weredetermined in duplicate on Vero cells incubated at 32° C. as describedabove. The limit of virus detection was 100 and 50 pfu/g for the NT andlung specimens, respectively.

Replication of CPD viruses was evaluated in the upper and lowerrespiratory tract of six-week-old Golden Syrian hamsters andimmunogenicity was also investigated. On day 0, groups of 18 hamsterswere inoculated intranasally under methoxyflurane anesthesia with 10⁶pfu of wt rRSV, Min_L, M2-1[A73S], M2-1[N88K] or NPM2-1[N88K]L.

On day 3, which corresponds to the peak of replication of wt rRSV inhamsters, 9 hamsters from each group were sacrificed by carbon dioxideinhalation. NT and lung tissue were harvested and homogenized asdescribed above. Virus titers were determined in duplicate on Vero cellsincubated at 32° C. as described above. The limit of virus detection was50 pfu/g in the NTs and lungs.

Two days before immunization and on day 26 post-immunization, blood fromnine hamsters per group was collected for serum collection and tomeasure of RSV antibody titers. On day 31, the hamsters were challengedby intranasal administration of 10⁶ pfu of wt rRSV. Three days afterchallenge the hamsters were sacrificed by carbon dioxide inhalation. NTand lung tissue were harvested and wt rRSV titers were determined induplicate on Vero cells incubated at 32° C. as described above.

Molecular dynamics analysis of M2-1 tetramer. Mutations were introducedto the crystal structure of the transcription antiterminator M2-1protein of human RSV (PDB ID 4C3D) using the SYBYL program (Certara).Molecular dynamics simulations were performed using the NAMD program(v.2.9).

Mutations were made to the crystal structure of the transcriptionantiterminator M2-1 protein of human RSV (PDB ID 4C3D) using the SYBYLprogram (Certara, St. Louis, Mo.). Mutants or wt RSV M2-1 wereexplicitly solvated with TIP3P water molecules and Na⁺ and Cl⁻counterions using the VMD program. All atom, isobaric-isothermal (1 atm,310 K) molecular dynamics simulations were performed with periodicboundary conditions using the NAMD program (v.2.9) on the Biowulf Linuxcluster at the National Institutes of Health, Bethesda, Md. afterexplicitly solvating and energy minimizing followed by warming to 310 Kin 10 K increments. Electrostatic interactions were calculated using theParticle-Mesh Ewald summation. The CHARMM27 force field was used withCHARMM atom types and charges. For all simulations, a 2 fsec integrationtime step was used along with a 12A cutoff. Langevin dynamics were usedto maintain temperature at 310 K and a modified Nosé-Hoover Langevinpiston was used to control pressure. Simulations were run for 100 nsec.

Statistical analysis. Distribution of the plaques sizes were analyzedusing Kolmogorov-Smirnov test followed by Bonferroni correction. Virusreplication and antibody responses in the animal experiments wereanalyzed using the nonparametric Kruskal-Wallis test with Dunn's posthoc analysis. A log10 transformation was applied to data sets whennecessary to obtain equal standard deviation among groups. Statisticswere performed using Prism 6 (GraphPad Software). Data were onlyconsidered significant at p<0.05.

Example 1 Generation of the Min_L and Min_FLC RSV Constructs

The design of the CPD RSV genes and the construction and rescue of Min_Land Min_FLC have been described previously in U.S. published applicationUS 2015-0368622 and in Le Nouen et al. (2014). Briefly, previouslydescribed computational algorithms (Coleman et al. 2008 and Mueller etal. 2010) were used to design CPD ORFs based on the RSV strain A2. Min_Lcontains the CPD L ORF, which exhibits 1,378 silent mutations comparedto wild-type (wt) L ORF. Min_FLC (for full-length clone) contained allCPD ORFs except M2-1 and M2-2, which were kept unmodified because theseoverlapping ORFs engage in coupled stop-start translation that dependson sequence (and possibly secondary structure) that is presentlyincompletely defined. Min_FLC contains 2,692 silent mutations comparedto wt RSV (FIG. 1, panel (A)). The amino acid sequence of Min_L andMin_FLC is identical to that of wt RSV. The viruses were constructedusing the RSV 6120 backbone, which has a 112-nt deletion in thedownstream NTR of the SH gene and 5 silent nucleotide point mutationsinvolving the last three codons and termination codon of the SH ORF.These changes in the SH gene stabilized the RSV cDNA during propagationin E. coli (Bukreyev et al. 2004). Wt RSV in this study was the 6120virus. Min_L and Min_FLC virus stocks were completely sequenced bySanger and Ion Torrent deep sequencing and found free of adventitiousmutations. The nucleotide sequence of Min_FLC is presented in SEQ IDNO:12 and that of Min_L is presented in SEQ ID NO:13.

Example 2 Codon-Pair Deoptimization (CPD) of Multiple RSV Genes Yieldeda Very Stable Temperature Sensitive (Ts) Phenotype Restricted toReplication at 32-34° C.

As mentioned above, Min_FLC (for full-length clone) is a mutant in which9 of the 11 RSV ORFs (excepting only M2-1 and M2-2) were CPD, resultingin a total of 2,692 silent mutations (FIG. 1, panel (A)). Min_FLC ishighly temperature-sensitive, with a shut-off temperature (T_(SH)) of35° C. for plaque formation, whereas wild-type (wt) rRSV readily formsplaques at 40° C. T_(SH) is defined as the lowest restrictivetemperature at which the difference in titer compared to that at 32° C.is reduced ≥100-fold compared to the difference in titer of wt rRSV atthe two temperatures.

To investigate Min_FLC stability, a temperature stress test wasemployed, representing a surrogate model for genetic stability duringvirus replication and spread from the cooler upper to the warmer lowerrespiratory tract. Ten independent 25-cm² replicate flasks of Vero cellswere infected with an initial MOI of 0.1 plaque forming unit (pfu)/cellof Min_FLC and subjected to serial passage at progressively increasingtemperatures for a total of 18 passage stages, representing 7 months ofcontinuous culture. (The flasks were incubated at the indicated startingtemperatures until extensive cytopathology was observed. Viruses wereharvested, and serially passed at increasingly restrictive temperatures(1° C. temperature increase, every other passage).) Two additionalreplicate flasks were infected and passaged in parallel at thepermissive temperature of 32° C. as controls (FIG. 1, panels (B) & (C)).One ml (out of a total of 5 ml) of the supernatant was used to inoculatethe next passage. After each passage, aliquots were frozen for titrationand sequence analysis by Sanger sequencing and/or deep sequencing asindicated. Virus titers were determined by plaque assay at thepermissive temperature (32° C.).

At 32° C., Min_FLC replicated consistently to titers of 10⁶ to 10⁷pfu/ml (FIG. 1, panel (B)). Deep sequencing of the complete genomes ofthe two control lineages after 18 passages revealed only low-level,sporadic mutations (FIG. 7), showing that Min_FLC was genetically stableunder permissive conditions. In the flasks incubated at increasingtemperature, Min_FLC replicated efficiently at 32 and 33° C. (10⁶ to 10⁷pfu/ml, FIG. 1, panel (C)). However, after the first passage at 34° C.(P5), virus replication was reduced 200-fold in all 10 lineages, and atthe end of the second passage at 35° C. (P8), virus was undetectable in9 lineages. In the 10th lineage, no virus was detected at the end of thefirst passage at 37° C. (P11). In contrast, as noted, wt rRSV exhibitsno growth restriction at temperatures up to at least 40° C.

Thus, Min_FLC was highly restricted, if not inactive at temperaturesabove 34-35° C. (the latter being its T_(SH)). Consequently, Min_FLCcannot escape its Ts phenotype and is phenotypically stable under stressconditions. Sequencing was not performed on Min_FLC specimens passedunder increasingly restrictive temperatures due to the rapid decrease intiters. These results fulfilled the expectation of phenotypic stabilityfor a CPD virus.

Example 3 Temperature Stress on the Min_L Virus Promoted the Emergenceof Multiple Mutations in Multiple Genes

The Min_L virus in which the L ORF alone (representing 48% of theaggregate RSV ORFs) was CPD, resulting in 1,378 silent mutations (51% asmany changes as in Min_FLC). Min_L has a T_(SH) of 37° C. Ten replicateflasks were infected with Min_L and passaged serially at progressivelyincreasing temperatures for a total of 8 passages, corresponding to 2months of continuous culture, and 2 additional replicate flasks wereinfected and passaged in parallel at 32° C. as controls (FIG. 1, panels(D) & (E)).

As expected, Min_L replicated efficiently (10⁷ pfu/ml) at each passageat 32° C. (FIG. 1, panel (D)). Sequence analysis of RNA from the controllineages at P6 by deep sequencing (FIG. 8) and at P8 by Sangersequencing (data not shown) revealed only sporadic, low-level mutations.In the 10 lineages passaged at increasing temperature, the titers ofMin_L in 9 flasks was decreased by about 20-fold at the end of P1 (37°C.) (FIG. 1, panel (E)). However, during the second passage at 37° C.,titers in the same 9 lineages increased by about 200-fold, suggestingthat selection and outgrowth of temperature-adapted mutants was alreadyoccurring. Following P3 (38° C.), virus titers in all 10 lineagesdecreased steadily: at P8 (second passage at 40° C.), virus wasundetectable in 7 lineages, whereas in 2 other lineages, titers werevery low (20 pfu/ml each). The remaining lineage (#3, colored in green)had a titer of 500 pfu/ml. Thus, the various Min_L lineages appeared toundergo a partial loss of the temperature-sensitivity phenotype, butultimately were strongly restricted at 40° C.

For each of the 10 lineages passaged at increasing temperature,whole-genome deep sequencing was performed at the end of P6 (the secondpassage at 39° C.), when virus replication was still detectable in eachlineage. Mutations present in ≥45% of the sequencing reads are shown inTable 1. Remarkably, many of these prominent mutations were in genes notsubjected to CPD. Specifically, of these 23 prominent mutations, 21 weredistributed among 6 ORFs (P, M, SH, G, M2-1 and L) and 2 were inextragenic regions. Of the 23 mutations, 11 (48%) and 5 (22%) occurredin the M2-1 and L ORFs, respectively. Of the 21 mutations present inORFs, all but one were missense mutations, suggesting a bias for aminoacid change. This positive selection for amino acid change suggests thatat least part of the adaptation of Min_L to selective stress involvedchanges in structure/function in various viral proteins. Some mutationswere common to several lineages. Specifically, the mutation [A73S] inthe anti-termination transcription factor M2-1 was prominent in 8 out ofthe 10 lineages. Another M2-1 mutation (N88K) and a mutation in L(A1479T) were prominent in 2 lineages. M2-1 was the only gene to haveone or more prominent mutations in every lineage.

Table S1 shows mutations that were present in ≥5% of the reads from theP6 specimens from the same experiment. With this lower cut-off, manymore mutations were evident in every gene except NS2. Similar to theprominent mutations that were shown in Table 1, these less prominentmutations were mostly missense mutations. In the CPD L ORF, only 17 outof the total of 31 mutations (55%) involved ant or a codon that had beenmodified during CPD (Table S1).

Whole-genome deep sequencing analysis was performed to evaluate thetemporal appearance of mutations in the full passage series of lineages#3 and #8, which were of interest because they maintained the highesttiters during the stress test (FIG. 1, panel (E)) and thus have thegreatest de-attenuation. The appearance and frequency of the moreabundant mutations are shown graphically in FIG. 1, panels (F) (lineage#3) and (G) (lineage #8). A more detailed listing of the mutations isshown in Tables S2 and S3.

In both lineages, a single mutation ([A73S] in M2-1) appeared at P1 (13%of each lineage) and then increased at P2 (37 and 51% in lineage #3 and8, respectively). From P2, the two lineages went into differentevolutionary trajectories. In lineage #3, between P2 and P3, while thefrequency of M2-1 mutation [A73S] started to decline (30%), 10 othermutations in M2-1 appeared and constituted approximately 15 to 30% ofthe population (Table S2). One of these M2-1 mutations, namely [N88K],became abundant (71%) in P4, closely concurrent with an equally abundant(66%) mutation [E114V] in P (FIG. 1, panel (F). The other M2-1 mutationsdeclined and were undetectable beyond P5, suggestive of a selectivesweep. Two additional prominent mutations were acquired at P6 (N[K136R])and P7 (L[T1166I]). In lineage #8, mutation [A73S] in M2-1 was fixed atP4 (88%). At P2, two additional mutations (in the 5′ trailer region andin L) were acquired and became prominent and fixed by the end of P4.After the first passage at 40° C. (P7), some additional mutations wereacquired, three of which became prominent by the end of P8; one silentin L, one silent in N, and one in P[E113G].

Example 4 The Two Mutations N88K and A73S in the Anti-TerminationTranscription Factor M2-1 are Prominent but Incompatible

All 10 lineages at P6 had either M2-1 mutation [A73S] or [N88K] (Table1, FIG. 2, panel (A)). Thus, these 2 M2-1 mutations seemed to segregate.In addition, the disappearance of the [A73S] mutation during passageseries of lineage #3 coincided with the appearance and increase of[N88K], until the latter was present in the complete population (FIG. 1,panel (F)). The deep sequencing results of lineage #3 were re-evaluated,scoring only those reads that spanned both position 73 and 88 in M2-1,thus providing a linkage analysis. At P3 and P4, only 1% of the readshad both mutations (FIG. 2, panel (B)), suggesting that these twomutations in M2-1 are incompatible in the same genome and thusconstitute 2 separate virus populations.

To further characterize the dynamics of the main virus populations inlineage #3, linkage of the major mutations that appeared during thefirst 4 passages was investigated using PacBio long read, singlemolecule sequencing, which provided complete reads of an entire 8.2 kbregion from the 3′ genome end to the middle of the M2-2 ORF. This showedthat the first 4 passages contained four major virus subpopulations(FIG. 2, panel (C)). One was the original Min_L virus, whichprogressively decreased with passage. Another subpopulation that carriedthe M2-1 mutation [A73S] alone peaked at P2 and almost disappeared inP4. Another carried 7 mutations in M2-1 (3 synonymous, 4 non-synonymous)that appeared together at P2, reached a maximum at P3 (about 20%) andthen disappeared. Finally, the fourth subpopulation contained theP[E114V] and M2-1[N88K] mutations that appeared together at P3 andbecame prominent at P4.

Example 5 Introduction of the Mutation(s) N[K136R], P[E114V],M2-1[N88K], M2-1[A73S] and L[T1166I] into Min_L

Direct identification of mutation(s) responsible for the loss oftemperature sensitivity of Min_L was investigated by introducing intoMin_L, individually and in combinations, major mutations that had beenidentified in lineage #3, namely N[K136R], P[E114V], M2-1[N88K], andL[T1166I] (FIG. 1, panel (F)), as well as the M2-1 mutation [A73S] thatwas one of the prominent mutations in replicate #8 (FIG. 1, panel (G)).The resulting 12 viruses (FIG. 3, panel (A)) were recovered andsequenced completely, confirming the correct sequences and absence offurther mutations.

This was performed using the Quickchange Lightning Site-directedMutagenesis kit (Agilent) following the manufacturer's recommendations.cDNAs were completely sequenced by Sanger sequencing using a set ofspecific primers. CPD viruses with targeted mutations were then rescuedfrom cDNA as described previously. Briefly, BSR T7/5 cells weretransfected using Lipofectamine 2000 (Life technologies) and a plasmidmixture containing 5 μg of full-length cDNA, 2 μg each of pTM1-N andpTM1-P, and 1 μg each of pTM1-M2-1 and pTM1-L. After overnightincubation at 37° C., transfected cells were harvested by scraping intomedia, added to sub-confluent monolayers of Vero cells, and incubated at32° C. The rescued viruses were harvested between 11 and 14 dayspost-transfection.

The introduction of the N[K136R] or P[E114V] mutation alone conferredapproximately a 1° C. increase in TSH (FIG. 3, panel (B)) compared withMin_L, whereas L[T1166I] alone did not have an effect. Interestingly,the introduction of M2-1[A73S] or [N88K] alone induced a 2° C. increasein T_(SH), suggesting that either of these two M2-1 mutations aloneplayed the greatest role in the de-attenuation of Min_L. The combinationof the N or P mutation with M2-1[N88K] conferred a further, smallincrease in T_(SH) (average of 2.5° C. from three independentexperiments). The combination of the N, P, and M2-1[N88K] mutationsinduced a 3° C. increase in TSH (40° C.) compared to Min_L, which wasnot further increased by the addition of the L mutation. Thisillustrated the additive role of the N, P, and M2-1[N88K] mutations inthe increase in the T_(SH) of lineage #3. The combination of M2-1[A73S]and [N88K] did not confer any increase in the TSH of Min_L, illustratingtheir incompatibility, as predicted based on the deep sequencingresults.

The effects of these mutations on the kinetics and efficiency of Min_Lreplication in Vero cells was also studied (FIG. 3, panels (C) & (D)).The effects of the mutations were more evident at 37° C. (FIG. 3, panels(C), (D), right panels) than at 32° C. (left panels), as would beexpected for temperature-sensitivity mutations. The N and P mutationsalone and in combination had only a small effect on increasing viralreplication compared to Min_L. In contrast, the introduction of eitherthe M2-1[N88K] or the [A73S] mutation alone resulted in a substantialincrease in replication, and this was not much affected by the furtheraddition of the N, P, and L mutations. In addition, virus bearing bothof the incompatible M2-1[A73S] and [N88K] mutations replicated similarto or less efficiently than Min_L at 37° C. and 32° C., respectively(FIG. 3, panel (D), left and right panels). Thus, the M2-1[N88K] or[A73S] mutations played the major role in restoring the ability of Min_Lto replicate in Vero cells, but they were incompatible.

Further, the effects of the introduced mutations on the kinetics ofviral gene transcription, viral genomic RNA synthesis, proteinexpression, and virus particle production in a single infection cycle(FIG. 4, panels (A) to (E)) was investigated. Vero cells were infectedat an MOI of 3 pfu/cell with the indicated viruses, and samples werecollected for analysis every 4 h for 24 h.

Analysis of the accumulation of the 9 smaller RSV mRNAs (i.e., allexcept L) was performed by positive-sense-specific RT-qPCR assaysspecific for each mRNA. Data for the P mRNA, which are generallyrepresentative, are shown in FIG. 4, panel (A), and the complete dataset for these 9 mRNAs is shown in FIG. 9. In general, transcription wasgreatly reduced at 37° C. for Min_L compared to wt rRSV. Theintroduction of either M2-1 mutation into Min_L resulted in asubstantial restoration of transcription. The further addition of the N,P, and L mutations to M2-1[N88K] provided a further modest, but mostlyconsistent, increase. Western blot analysis showed that, as expected,the viral protein accumulation occurred later than that of the mRNAs butotherwise the pattern was similar to the mRNA accumulation (FIG. 4,panel (B) and FIG. 10).

The accumulation of the RSV L mRNA by positive-sense-specific,L-specific RT-qPCR (FIG. 4, panel (C)) was examined. At 32° C., a basallevel of L mRNA was detected in Min_L-infected cells, but there wasessentially no increase with time, in contrast to the progressiveincrease with time observed with wt L mRNA. The extensive sequencedifferences in the wt and CPD L genes necessitated the use of differentprimer pairs for wt rRSV versus Min_L derivatives, precluding directcomparison of relative abundances at the different time points. At 37°C., CPD L mRNA was undetectable, indicating a strong restriction at thistemperature. The addition of the M2-1[N88K] or [A73S] mutation to Min_Lpartly restored CPD L gene transcription at both 32 and 37° C. Theadditional inclusion of the N, P, and L mutations further increased Lgene expression.

The production of cell-associated genomic RNA (FIG. 4, panel (D)) byMin_L was almost undetectable at either 32 or 37° C. but was detected at24 hpi at both 32 and 37° C. by M2-1[A73S] and M2-1[N88K], and infurther increased amounts in NPM2-1[N88K]L infected cells. Genomic RNAproduction by wt rRSV was detectable starting at 12 hpi at both 32 and37° C. and was higher compared to NPM2-1[N88K]L.

The production of infectious virus particles was concurrent with theaccumulation of genomic RNA (FIG. 4, panel (E)). At 32° C., Min_L virustiters started to increase only at 24 hpi, while no increase wasdetected at 37° C. M2-1[A73S] and M2-1[N88K] virus particles started toaccumulate earlier (20 hpi at both temperatures) and at higher levels(6-and 110-fold higher at 32 and 37° C., respectively) than Min_Lparticles. NPM2-1[N88K]L virus production was first detected at 16 hpiat both temperatures and also at greater amounts (9 and 300-fold higherat 32 and 37° C., respectively) than Min_L virus production. Infectiouswt rRSV was first observed at 12 hpi at both temperatures (FIG. 4, panel(E)), at higher level than NPM2-1[N88K]L (10-fold higher at both 32 and37° C.).

The plaque sizes produced in Vero cells were measured, as an additionalparameter for virus fitness (FIG. 4, panels (F) & (G)). Wt rRSV producedplaques of significantly larger size than Min_L (p<0.05). Addition ofthe M2-1 [A73S] or [N88K] mutations to Min_L increased virus fitness,resulting in plaque sizes that were not significantly different fromthose of wt rRSV (p>0.05 compared with wt rRSV). Plaques induced byM2-1[A73S][N88K] were smaller than Min_L plaques, further confirmingthat these two M2-1 mutations are incompatible.

Thus, the two most prominent mutations acquired under stress were twomissense mutations ([A73S] and [N88K]) in the M2-1 ORF, encoding the RSVtranscription anti-termination factor. Reintroduction of either of thesemutations by reverse genetics rescued a substantial part of thereplicative fitness of Min_L at 37° C., increasing viral genetranscription, protein expression, particle production, and plaque size.These two M2-1 mutations partly restored the transcription of the CPD Lgene at 37° C., which otherwise was below the level of detection at thistemperature. The partial restoration of L gene expression would beexpected to increase the production of the polymerase, although that wasnot directly monitored here due to its low abundance and a lack ofavailable antibody. We presume that an increase in the production of Lprotein would then increase transcription of all of the RSV genes,indirectly increase the synthesis of viral proteins, increase RNAreplication, and ultimately indirectly increase the production ofprogeny virus. These effects on the accumulation of viral mRNAs,proteins, genomic RNA, and progeny virions indeed were observed. Thus,the acquisition of either of two mutations in M2-1 adapted Min_L at 37°C., by increasing transcription of the CPD L gene.

The mechanism(s) behind the rescued CPD L gene expression by the twoM2-1 mutations remains unknown. The RSV M2-1 protein is necessary forthe efficient synthesis of full-length mRNAs, which otherwise terminateprematurely. The M2-1 protein also increases the synthesis ofpolycistronic read-through mRNAs. It likely binds nascent mRNAco-transcriptionally and prevents termination by the viral polymerase.In addition, the M2-1 protein binds directly to P. The binding of P andRNA to M2-1 was found to be mutually exclusive due to partiallyoverlapping interaction surfaces. Although A73 and N88 are away from theRNA/P binding interface, they could possibly be on the path of theexiting nascent RNA molecule. A simple model would be that the 1,378 ntchanges that were introduced during CPD affected the L gene template soas to reduce the efficiency of transcription elongation of the nascent LmRNA. L transcription was partly restored by the M2-1 mutations throughsome effect on the polymerase complex. The prominent mutations that wereacquired under stress were most frequent in the M2-1 ORF, but also werefound in P, N, and L ORFs, all of which encode viral proteins involvedin RNA synthesis. These additional N, P and L mutations furtherincreased the efficiency of CPD L gene transcription possibly by alsoincreasing the efficiency of transcription elongation on the CPD L gene.

Example 6 Computer-Based Molecular Dynamics Simulations (MDS)

Computer-based molecular dynamics simulations (MDS) was used toinvestigate possible effects of the M2-1 [A73S] and [N88K] mutations onM2-1 structure (FIG. 6). The M2-1 tetramer is shown in FIG. 6, panel (A)with specific views in panels (B), (C), and (D). In the wt M2-1tetramer, a salt bridge is predicted to exist between K19 of one monomerand D116 of the adjoining monomer. These amino acids are shown for thered and cyan monomers (FIG. 6, panel (B)). MDS suggests that the saltbridge helps stabilize the interaction between adjacent monomers. TheA73 residue of a third monomer is predicted to be in close proximity butnot involved in interactions. When A73 is changed to serine ([A73S],FIG. 6, panel (C)), the salt bridge between K19 and D116 is predicted tobe maintained. In addition, unlike the alanine, a serine at codon 73 ispredicted to form a hydrogen bond with K19 and in some MDS time frames ahydrogen bond with D116 (not shown). Thus, S73 could provide newstabilizing links between each adjoining monomers. The predicted effectof the N88K mutation is to increase stability within rather than betweenmonomers. Specifically, in the wt M2-1 tetramer structure, N88 ispredicted to form a hydrogen bond with S82 (FIG. 6, panel (B)). Incontrast, a lysine residue at codon 88 is predicted to form anintra-monomer salt-bridge with E70 (FIG. 6, panel (D)). The K88 would nolonger interact with S82. In addition, the hydrophobic carbon chain ofK88 is predicted to form a number of intra-monomer van der Waalsinteractions with L74. Thus, the prominent M2-1 mutations acquiredduring the stress test are predicted to create new interactions between(A73S) and within (N88K) M2-1 monomers. This increased stabilitypresumably could contribute to rescue transcription of the CPD L gene.Interestingly, this increased stability is not expected to be maintainedwhen both mutations are present together. Indeed, these two mutationscould possibly form an H-bonded pair between the side chains of the S73and K88 which would result in less flexibility of the loop on which K88resides. This reduced flexibility could explain the incompatibility ofthese 2 mutations.

Interestingly, mutations that were found in P ([E113G] and [E114V]) arelocalized in the interaction domain of P with M2-1. Mutations at these 2positions were shown to increase the affinity of P for M2-1. This workfurther supports the theory that the compensatory mutations act byincreasing the stability of the ribonucleoprotein complex, which wehypothesize may facilitate transcription of the CPD L gene.

As mentioned, a single mutation in the M2-1 gene (A73S) that appeared inthe first passage of Min_L at 37° C. and was found in 8 of 10 cultureswas sufficient to rescue Min_L replication at that temperature. Inaddition, this single mutation conferred increased replication to Min_Lin hamsters. We had anticipated that de-attenuation of a CPD ORF wouldinvolve multiple changes in the CPD sequence conferring incrementalde-attenuation. However, this study shows that a single mutation in adifferent gene was sufficient to yield substantial de-attenuation.Therefore deoptimization involving large numbers of nt changes does notnecessarily provide a stable attenuation phenotype.

Example 7 Introduction of De-Attenuating Mutations from Min_L intoMin_FLC

The major mutations that were introduced into Min_L, namely N[K136R],P[E114V], M2-1[N88K], M2-1[A735], and L[T1166I], were introduced invarious combinations into Min_FLC and assessed for virus titer followingrecovery (FIG. 11, panel (A)) and T_(SH) (FIG. 11, panel (B)). TheM2-1[N88K] and [A73S] mutations individually did not increase thefitness of Min_FLC as measured by viral titer or T_(SH). The combinationof the N, P, and M2-1[N88K] mutations conferred a 2° C. increase in TsH,but this virus only grew to a low titer.

Surprisingly, the introduction of the L[T1166I] mutation into Min_FLCalone or in combinations with one or more of the other mutationsappeared to inhibit recovery. Thus, none of these mutations improved theoverall fitness of Min_FLC, even though it bears the same CPD L gene asMin_L. This result suggests that multiple CPD ORFs augment phenotypicstability under selective pressure.

Example 8 Evaluation of Min_L Derivatives in Mice and Hamsters

The replication of the Min_L derivatives was evaluated in vivo (FIG. 5).BALB/c mice were infected intranasally (IN) with 10⁶ pfu of each virus.Nasal turbinates (NT) and lungs were harvested on days 4 (n=8 pervirus), 5 (n=8), and 10 (n=4) post-infection (pi). At the peak of virusreplication (day 5 pi; FIG. 5, panel (B)), virus was detected in the NTof only 2 mice infected with Min_L, and 3 mice infected with M2-1[N88K].Replication of M2-1[A73S] was detected in 4 of 8 mice, which wascomparable to wt rRSV. NPM2-1[N88K]L replication was not detected in theNT of any of the mice. In the lungs on day 5, replication of M2-1[N88K]and M2-1[A73S] was slightly reduced compared with Min_L, but was notstatistically different, and replication of NPM2-1[N88K]L was stronglyreduced in the lungs compared to Min_L. The day 10 titers are not shownbecause virus was recovered only from 2 animals, in the M2-1[A73S] groupat trace levels.

The same set of viruses was compared in hamsters (FIG. 5, panel (C)). Onday 3, NT and lungs were harvested from 9 hamsters per virus. In the NT,Min_L replication was reduced approximately 100-fold compared to wt rRSV(p<0.01). Replication of M2-1[N88K] was modestly increased compared toMin_L, but remained significantly attenuated compared to wt rRSV. Incontrast, the titers of M2-1[A73S] were further increased compared toMin_L, and were not statistically different from wt rRSV. Interestingly,replication of NPM2-1 [N88K]L in the NTs was reduced compared to Min_L.In the lungs, Min_L and M2-1 [N88K] were detected in only 1 out of 9hamsters for each virus, and replication of NPM2-1 [N88K]L wasundetectable. In contrast, replication of M2-1 [A73S] was increasedcompared to Min_L, as 5 out of 9 hamsters exhibited virus replication toabout 10² pfu/g. Thus, in hamsters, the mutation M2-1[A73S] increasedthe replication of Min_L, a marker of de-attenuation, while theM2-1[N88K] mutation did not affect the replication of Min_L, and thecombination of the N, P, L, and M2-1[N88K] mutations decreasedreplication.

Despite a significant restriction of replication, Min_L and theMin_L-derived viruses induced titers of antibodies that were notstatistically different from those induced by wt rRSV (FIG. 5, panel(D)). The M2-1 [A73S] virus induced significantly higher levels ofRSV-neutralizing serum antibodies than Min_L and M21-1[N88K].Interestingly, the NPM2-1 [N88K]L virus also was comparable to wt rRSVin inducing RSV-neutralizing antibodies despite its highly restrictedreplication. On day 31, hamsters were challenged IN with wt rRSV, and NTand lungs were harvested 3 days post-challenge. No detectable challengevirus replication was detected except for a trace of virus in one animalin the Min_L group (not shown).

Example 9 Genetic Stability of the Min_L-NPM2-1[N88K]L Virus

The observation that the NPM2-1 [N88K]L virus was more highly attenuatedthan Min_L and yet was as immunogenic as wt rRSV identified this virusas a promising vaccine candidate. Therefore, its stability was evaluatedin a temperature stress test involving 4 passages at 39° C. and 4passages at 40° C., corresponding to 2 months of continuous passage(FIG. 12). Sanger sequencing of the complete genome of the final passageof the 10 different stressed lineages and the 2 control flasks did notdetect any abundant mutations (not shown). This showed that introductionof the N, P, M2-1 [N88K], and L mutations into Min_L to create thepromising NPM2-1[N88K]L virus conferred genetic stability. Thenucleotide sequence of Min_L-NPM2-1[N88K]L is shown in FIG. 14 andrepresented by SEQ ID NO: 14.

TABLE 1 Mutations detected in individual lineages of Min_L at the end ofP6 (second passage at 39° C.) of the temperature stress test, present at≥45% frequency^(a). Percentage of reads with mutation in Nt indicatedlineage number^(a) Gene mutation Aa mutation 1 2 3 4 5 6 7 8 9 10Intergenic g1123a — 85 NS2-N P a2687u E114V 96 M u3798a N179K 61 SHc4369a H22Q 81 SH c4387g I28M 71 G a5384g E232 (silent) 47 M2-1 g7823uA73S 99 93 61 48 83 63 87 57 M2-1 c7870a N88K 94 96 M2-1 a8013g E136G 48L u10548c^(b) Y684H^(b) 97 L u10797c^(b, c) S767P^(b, c) 82 L g12933aA1479T 63 85 L a13783c^(b) Y1762S^(b) 83 5′ extragenic u15100c — 75(trailer) ^(a)Percentage of reads with the indicated mutation; onlymutations present in ≥45% of the reads are shown. Nucleotide numberingis based on RSV sequence M74568 (biological wt RSV strain A2). Mutationspresent in ≥5% of reads from this same experiment are shown in Table S1.^(b)Mutation involving a codon that had been changed as part of CPD ofthe L ORF. ^(c)Mutation involving a nucleotide position that had beenchanged as part of CPD of the L ORF.

TABLE S1 Mutations detected (at a frequency of ≥5%) in each of the 10lineages of Min_L at the end of P6 (second passage at 39° C.) of thetemperature stress test^(a). Lineage number and the percentage of readsNt with the indicated mutation^(a) Gene mutation Aa mutation 1 2 3 4 5 67 8 9 10 NS1 g101a M1I 26 NS1 c439a S114Y 6 10 9 12 NS1 a441c K115Q 2519 32 Intergenic g1104a — 7 NS2-N Intergenic g1120a — 10 NS2-NIntergenic g1123a — 11 85 NS2-N N a1547g K136R 32 N c1737u G199 (silent)8 N a2293g K385E 7 N a2295g K385 (silent) 19 P a2386g N14D 12 P u2434cS30P 12 P g2683a E113K 13 P a2687u/g E114V 96 34 P a2695c S117R 20 Pg2926a A194T 16 Intergenic g3167a — 10 18 P-M Intergenic g3191c — 23 1716 P-M M a3428g N56S 6 M u3798a N179K 61 M a3821u N187I 11 10 15 SHc4369a H22Q 9 11 36 10 26 81 12 SH c4387g I28M 71 G a5384g E232 (silent)47 G g5499a E271K 18 Intergenic u5646a / 11 G-F F u5755a F32I 26 Fg6115a V152I 5 F g6382u A241S 30 F g6425a S255N 6 F u7298c L546P 7 Fg7330u V557F 7 F a7381u N574Y 10 Intergenic c7552a — 5 F-M2 M2-1 c7807gD67E 14 M2-1 g7823u A73S 99 93 61 48 83 63 87 57 M2-1 u7852c S82(silent) 16 M2-1 u7855c Y83 (silent) 11 5 M2-1 u7857c I84T 11 12 14 M2-1u7866c I87T 23 8 15 23 M2-1 u7866a I87K 11 M2-1 c7870a N88K 94 13 96M2-1 a7872c N89T 30 M2-1 u7873c N89 (silent) 18 M2-1 a8013g E136G 21 3948 19 M2-2 u8255c N32 (silent) 11 M2-2 c8268u L37 (silent) 5 M2-2 c8428aS90Stop 5 L gene a8494g — 6 start L a8514g N6D 11 L u8563a^(b) V22E^(b)8 6 L u8950c^(b) V151A^(b) 32 L g8985a^(b) A163T^(b) 12 L a9560g K354(silent) 15 L c10029u^(b, c) R511C^(b, c) 14 L c10298g^(b, c)C600W^(b, c) 6 L a10301g V601 (silent) 7 8 L a10527u I677L 6 Lu10548c^(b) Y684H^(b) 97 L u10797c^(b, c) S767P^(b, c) 82 L a10972u^(b)E825V^(b) 5 L u11278a^(b) I927N^(b) 7 8 L g11535a^(b) V1013I^(b) 6 La11575g D1026G 21 L u11775g F1093V 6 L a11783g K1095 (silent) 8 7 6 Lc11790u Q1098Stop 6 L u11795c H1099 (silent) 5 L a11956g^(b) K1153R^(b)20 L a12078g M1194V 17 12 L g12114a V1206I 11 L a12210c^(b, c)S1238R^(b, c) 8 L u12386a D1296E 7 L g12933a A1479T 63 7 8 85 11 La13783c^(b) Y1762S^(b) 83 L u14045c^(b, c) I1849^(b, c) (silent) 25 Lc14204g^(b, c) Y1902Stop^(b, c) 6 L c14411u^(b, c) I1971^(b, c) (silent)18 L c14805u H2103Y 36 L c14834u^(b, c, d) H2112^(b, c, d) (silent) 6 5′extragenic u15100c / 75 5′ extragenic a15143g / 20 ^(a)Percentage ofreads with the indicated mutation; only mutations present in ≥5% of thereads are shown. Mutations detected in ≥50% of the reads are highlightedin yellow and mutations detected in 25 to 49% of reads are highlightedin green. Nucleotide numbering is based on RSV sequence M74568.^(b)Mutations involving a codon that had been changed as part of CPD ofL. ^(c)Mutations involving a nucleotide that had been changed as part ofCPD of L. ^(d)Mutation involving a nucleotide that had been changed aspart of CPD of L and that restored wt sequence.

TABLE S1-A Mutations detected (at a frequency of ≥25%) in each of the 10lineages of Min_L at the end of P6 (second passage at 39° C.) of thetemperature stress test^(a). Lineage number and the percentage of readsNt with the indicated mutation^(a) Gene mutation Aa mutation 1 2 3 4 5 67 8 9 10 NS1 g101a M1I 26 NS1 a441c K115Q 25 19 32 Intergenic g1123a —11 85 NS2-N N a1547g K136R 32 P a2687u/g E114V 96 34 M u3798a N179K 61SH c4369a H22Q 9 11 36 10 26 81 12 SH c4387g I28M 71 G a5384g E232(silent) 47 F u5755a F32I 26 F g6382u A241S 30 M2-1 g7823u A73S 99 93 6148 83 63 87 57 M2-1 c7870a N88K 94 13 96 M2-1 a7872c N89T 30 M2-1 a8013gE136G 21 39 48 19 L u8950c^(b) V151A^(b) 32 L u10548c^(b) Y684H^(b) 97 Lu10797c^(b, c) S767P^(b, c) 82 L g12933a A1479T 63 7 8 85 11 La13783c^(b) Y1762S^(b) 83 L u14045c^(b, c) I1849^(b, c) (silent) 25 Lc14805u H2103Y 36 5′ extragenic u15100c / 75 ^(a)Percentage of readswith the indicated mutation; only mutations present in ≥25% of the readsare shown. Nucleotide numbering is based on RSV sequence M74568.^(b)Mutations involving a codon that had been changed as part of CPD ofL. ^(c)Mutations involving a nucleotide that had been changed as part ofCPD of L.

TABLE S1-B Mutations detected (at a frequency of ≥50%) in each of the 10lineages of Min_L at the end of P6 (second passage at 39° C.) of thetemperature stress test^(a). Lineage number and the percentage of readsNt with the indicated mutation^(a) Gene mutation Aa mutation 1 2 3 4 5 67 8 9 10 Intergenic g1123a — 11 85 NS2-N P a2687u/g E114V 96 34 M u3798aN179K 61 SH c4369a H22Q 9 11 36 10 26 81 12 SH c4387g I28M 71 M2-1g7823u A73S 99 93 61 48 83 63 87 57 M2-1 c7870a N88K 94 13 96 Lu10548c^(b) Y684H^(b) 97 L u10797c^(b, c) S767P^(b, c) 82 L g12933aA1479T 63 7 8 85 11 L a13783c^(b) Y1762S^(b) 83 5′ extragenic u15100c /75 ^(a)Percentage of reads with the indicated mutation; only mutationspresent in ≥50% of the reads are shown. Nucleotide numbering is based onRSV sequence M74568. ^(b)Mutations involving a codon that had beenchanged as part of CPD of L. ^(c)Mutations involving a nucleotide thathad been changed as part of CPD of L.

TABLE S2 Accumulation of mutations in passages 1 to 8 (from 37 to 40°C.) in lineage #3 during the temperature stress test^(a). Passage (P)number (temperature) and the percentage of reads with the indicatedmutation Nt P1 P2 P3 P4 P5 P6 P7 P8 Gene mutation Aa mutation (37) (37)(38) (38) (39) (39) (40) (40) NS1 gene g45a / 22 17 start NS1 u308c N70(silent) 14 25 NS1 c439a S114Y 5 5 4 6 N a1547g K136R 33 67 66 P a2687uE114V 19 71 87 96 99 100 M a3281g K7R 9 15 M2-1 c7807g D67E 8 5 M2-1g7823u A73S 13 37 30 12 5 M2-1 u7833c V76A 7 5 M2-1 u7855c Y83 (silent)19 26 12 6 M2-1 u7866c I87T 21 29 14 7 M2-1 c7870a N88K 14 66 85 95 100100 M2-1 u7873c N89 (silent) 16 26 13 6 M2-1 u7875c I90T 19 26 12 5 M2-1u7879c T91 (silent) 14 22 10 4 M2-1 u7965c L120P 18 25 12 5 M2-1 u8011cI135 (silent) 19 27 12 6 L u8930c^(b, c) G144^(b, c) (silent) 16 23 Lu8950c^(b) V151A^(b) 17 24 L u10548c^(b) Y684H^(b) 4 10 12 20 L u10556cD686 (silent) 5 6 7 L u10562c^(b, c, d) Y688^(b, c) (silent) 7 14 10 Lu10569c^(b) Y691H^(b) 4 7 L u10571c^(b, c, d) Y691^(b, c, d) (silent) 69 10 L a10572g^(b) I692V^(b) 6 7 6 L c11995u^(b) T1166I^(b) 40 68 La12078g M1194V 15 19 8 9 L c12239u^(b, c, d) N1247^(b,c,d) (silent) 1012 L a13361c T1621 (silent) 5 5 ^(a)Percentage of reads with theindicated mutation; only mutations detected in at least 2 consecutivepassages with ≥5% of the reads in 1 passage are shown. The temperaturesof the specific passages are shown in parentheses. Mutations detected in≥50% of the reads at a given passage are highlighted in yellow andmutations detected in 25 to 49% of the reads are highlighted in green.Nucleotide numbering is based on RSV sequence M74568. ^(b)Mutationsinvolving a codon that had been changed as part of CPD of L.^(c)Mutations involving a nucleotide that had been changed as part ofCPD of L. ^(d)Mutations involving a nucleotide that had been changed aspart of CPD of L and that restored wt sequence.

TABLE S2-A Accumulation of mutations in passages 1 to 8 (from 37 to 40°C.) in lineage #3 during the temperature stress test^(a). Passage (P)number (temperature) and the percentage of reads with the indicatedmutation Nt P1 P2 P3 P4 P5 P6 P7 P8 Gene mutation Aa mutation (37) (37)(38) (38) (39) (39) (40) (40) NS1 u308c N70 (silent) 14 25 N a1547gK136R 33 67 66 P a2687u E114V 19 71 87 96 99 100 M2-1 g7823u A73S 13 3730 12 5 M2-1 u7855c Y83 (silent) 19 26 12 6 M2-1 u7866c I87T 21 29 14 7M2-1 c7870a N88K 14 66 85 95 100 100 M2-1 u7873c N89 (silent) 16 26 13 6M2-1 u7875c I90T 19 26 12 5 M2-1 u7965c L120P 18 25 12 5 M2-1 u8011cI135 (silent) 19 27 12 6 L c11995u^(b) T1166I^(b) 40 68 ^(a)Percentageof reads with the indicated mutation; only mutations detected in atleast 2 consecutive passages with ≥25% of the reads in 1 passage areshown. The temperatures of the specific passages are shown inparentheses. Nucleotide numbering is based on RSV sequence M74568.bMutations involving a codon that had been changed as part of CPD of L.^(c)Mutations involving a nucleotide that had been changed as part ofCPD of L. ^(d)Mutations involving a nucleotide that had been changed aspart of CPD of L and that restored wt sequence.

TABLE S2-B Accumulation of mutations in passages 1 to 8 (from 37 to 40°C.) in lineage #3 during the temperature stress test^(a). Passage (P)number (temperature) and the percentage of reads with the indicatedmutation Nt P1 P2 P3 P4 P5 P6 P7 P8 Gene mutation Aa mutation (37) (37)(38) (38) (39) (39) (40) (40) N a1547g K136R 33 67 66 P a2687u E114V 1971 87 96 99 100 M2-1 c7870a N88K 14 66 85 95 100 100 L c11995u^(b)T1166I^(b) 40 68 ^(a)Percentage of reads with the indicated mutation;only mutations detected in at least 2 consecutive passages with ≥50% ofthe reads in 1 passage are shown. The temperatures of the specificpassages are shown in parentheses. Nucleotide numbering is based on RSVsequence M74568. ^(b)Mutations involving a codon that had been changedas part of CPD of L.

TABLE S3 Accumulation of mutations in passages 1 to 8 (from 37 to 40°C.) in lineage #8 during the temperature stress test^(a). Passage (P)number (temperature) and the percentage of reads with the indicatedmutation Nt P1 P2 P3 P4 P5 P6 P7 P8 Gene mutation Aa mutation (37) (37)(38) (38) (39) (39) (40) (40) NS1 c439a S114Y 7 5 8 5 5 5 N u2127c A329(silent) 4 28 72 P a2684g E113G 33 61 Intergene a4282g / 6 44 M-SH SHgene a4625u / 18 end G a5170g N161S 10 G c5310u L208F 13 G u5541c Y284H5 34 F g5800a A47T 10 F u7298c L546P 5 9 8 4 M2-1 g7823u A73S 13 51 6988 88 86 94 100 M2-1 u7866c I87T 17 10 4 M2-1 u7866a I87K 5 10 5 M2-1u7875c I90T 17 9 M2-1 u7879c T91 (silent) 15 9 M2-1 u7927c N107 (silent)19 11 4 M2-2 u8279c N40 (silent) 14 8 M2-2 u8294c N45 (silent) 16 9 M2-2u8419c I87T 26 14 4 M2-2 gene u8466c / 24 14 4 end L c9156u^(b)Q220Stop^(b) 5 L a10434u M646L 8 43 L g10824u G776C 10 L a11363g I955M22 10 L g11535a^(b) V1013I^(b) 6 5 L a12033c I1179L 12 43 L g12933aA1479T 10 40 76 82 85 94 97 L a13527g N1677D 5 10 L c13670u^(b, c, d)N1724^(b, c, d) (silent) 7 L u13850c^(b, c, d) G1784^(b, c, d) (silent)5 L c14204g^(b, c) Y1902Stop^(b, c) 7 L c14411u^(b, c) I1971^(b, c)(silent) 6 15 11 17 11 L u14984c F2162 (silent) 44 94 5′ UTR u15100c /13 41 86 92 86 95 100 ^(a)Percentage of reads with the indicatedmutation; only mutations detected in at least 2 consecutive passageswith ≥5% of the reads in 1 passage are shown. The temperatures of thespecific passages are shown in parentheses. Mutations detected in ≥50%of the reads at a given passage are highlighted in yellow and mutationsdetected in 25 to 49% of the reads are highlighted in green. Nucleotidenumbering is based on RSV sequence M74568. ^(b)Mutations involving acodon that had been changed as part of CPD of L. ^(c)Mutations involvinga nucleotide that had been changed as part of CPD of L. ^(d)Mutationsinvolving a nucleotide that had been changed as part of CPD of L andthat restored wt sequence.

TABLE S3-A Accumulation of mutations in passages 1 to 8 (from 37 to 40°C.) in lineage #8 during the temperature stress test^(a). Passage (P)number (temperature) and the percentage of reads with the indicatedmutation Nt P1 P2 P3 P4 P5 P6 P7 P8 Gene mutation Aa mutation (37) (37)(38) (38) (39) (39) (40) (40) N u2127c A329 (silent) 4 28 72 P a2684gE113G 33 61 Intergene a4282g / 6 44 M-SH G u5541c Y284H 5 34 M2-1 g7823uA73S 13 51 69 88 88 86 94 100 M2-2 u8419c I87T 26 14 4 L a10434u M646L 843 L a12033c I1179L 12 43 L g12933a A1479T 10 40 76 82 85 94 97 Lu14984c F2162 (silent) 44 94 5′ UTR u15100c / 13 41 86 92 86 95 100^(a)Percentage of reads with the indicated mutation; only mutationsdetected in at least 2 consecutive passages with ≥25% of the reads in 1passage are shown. The temperatures of the specific passages are shownin parentheses. Nucleotide numbering is based on RSV sequence M74568.

TABLE S3-B Accumulation of mutations in passages 1 to 8 (from 37 to 40°C.) in lineage #8 during the temperature stress test^(a). Passage (P)number (temperature) and the percentage of reads with the indicatedmutation Nt P1 P2 P3 P4 P5 P6 P7 P8 Gene mutation Aa mutation (37) (37)(38) (38) (39) (39) (40) (40) N u2127c A329 (silent) 4 28 72 P a2684gE113G 33 61 M2-1 g7823u A73S 13 51 69 88 88 86 94 100 L g12933a A1479T10 40 76 82 85 94 97 L u14984c F2162 (silent) 44 94 5′ UTR u15100c / 1341 86 92 86 95 100 ^(a)Percentage of reads with the indicated mutation;only mutations detected in at least 2 consecutive passages with ≥50% ofthe reads in 1 passage are shown. The temperatures of the specificpassages are shown in parentheses. Nucleotide numbering is based on RSVsequence M74568.

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1. An isolated polynucleotide molecule encoding a recombinantrespiratory syncytial virus (RSV) variant having an attenuated phenotypecomprising a RSV genome or antigenome sequence, wherein the RSV genomeor antigenome is modified by a missense mutation in the L ORF at aposition corresponding to T1166 of the L protein in SEQ ID NO:11.
 2. Theisolated polynucleotide molecule of claim 1, wherein the missensemutation is a result of single nucleotide change of the second codonnucleotide for T1166 at position 11995 of the positive strand of RSVsequence M74568.
 3. The isolated polynucleotide molecule of claim 1,wherein the missense mutation is T1166I of the L protein in SEQ ID NO:11.
 4. The isolated polynucleotide molecule of claim 1, wherein the RSVgenome or antigenome is further modified by a missense mutation in theM2-1 ORF at a position corresponding to N88 of the M2-1 protein in SEQID NO:9, a missense mutation in the M2-1 ORF at a position correspondingto A73 of the M2-1 protein in SEQ ID NO:9, a missense mutation in the NORF at a position corresponding to K136 of the N protein in SEQ ID NO:3,or a missense mutation in the P ORF at a position corresponding to E114of the P protein in SEQ ID NO:4, or any combination thereof.
 5. Theisolated polynucleotide molecule of claim 4, wherein the missensemutation in the M2-1 ORF at a position corresponding to N88 of the M2-1protein in SEQ ID NO:9 is N88K, the missense mutation in the M2-1 ORF ata position corresponding to A73 of the M2-1 protein in SEQ ID NO:9 isA73S, the missense mutation in the N ORF at a position corresponding toK136 of the N protein in SEQ ID NO:3 is K136R, or the missense mutationin the P ORF at a position corresponding to E114 of the P protein in SEQID NO:4 is E114V, or any combination thereof.
 6. The isolatedpolynucleotide molecule of claim 1, wherein the RSV genome or antigenomeis further modified by two or more of a missense mutation in the M2-1ORF at a position corresponding to N88 of the M2-1 protein in SEQ IDNO:9, a missense mutation in the M2-1 ORF at a position corresponding toA73 of the M2-1 protein in SEQ ID NO:9, a missense mutation in the N ORFat a position corresponding to K136 of the N protein in SEQ ID NO:3, anda missense mutation in the P ORF at a position corresponding to E114 ofthe P protein in SEQ ID NO:4.
 7. The isolated polynucleotide molecule ofclaim 6, wherein the two or more missense mutations comprise a firstmissense mutation in a first RSV protein and a second missense mutationin a second RSV protein.
 8. The isolated polynucleotide molecule ofclaim 6, wherein the missense mutation in the M2-1 ORF at a positioncorresponding to N88 of the M2-1 protein in SEQ ID NO:9 is N88K, themissense mutation in the M2-1 ORF at a position corresponding to A73 ofthe M2-1 protein in SEQ ID NO:9 is A73S, the missense mutation in the NORF at a position corresponding to K136 of the N protein in SEQ ID NO:3is K136R, or the missense mutation in the P ORF at a positioncorresponding to E114 of the P protein in SEQ ID NO:4 is E114V, or anycombination thereof.
 9. The isolated polynucleotide molecule of claim 1,wherein the RSV genome or antigenome comprises a deletion of one or moreORF codons resulting in a deletion of one or more amino acid deletionsin at least one of the RSV proteins selected from M2-2, NS1 and NS2. 10.The isolated polynucleotide molecule of claim 1, wherein the RSV genomeor antigenome is codon-pair deoptimized.
 11. The isolated polynucleotidemolecule of claim 1, wherein the L ORF of the RSV genome or antigenomeis codon-pair deoptimized.
 12. A vector comprising the isolatedpolynucleotide molecule of claim
 1. 13. A cell comprising the isolatedpolynucleotide of claim
 1. 14. A pharmaceutical composition comprisingan immunologically effective amount of the recombinant RSV variantencoded by the isolated polynucleotide molecule of claim
 1. 15. A methodof vaccinating a subject against RSV comprising administering thepharmaceutical composition of claim
 14. 16. A method of inducing animmune response comprising administering the pharmaceutical compositionof claim
 14. 17. The method of claim 14, wherein the pharmaceuticalcomposition is administered intranasally.
 18. The method of claim 14,wherein the pharmaceutical composition is administered via injection,aerosol delivery, nasal spray, or nasal droplets, or any combinationthereof.
 19. A live attenuated RSV vaccine comprising the recombinantRSV variant encoded by the isolated polynucleotide of claim
 1. 20. Apharmaceutical composition comprising the RSV vaccine of claim 19.